JP2016101161A - Gene marker, method, reporter vector, primer set and assay kit for examining cellular property of test cells, and apparatus for examining cellular property - Google Patents

Abstract

To provide a means for evaluating cell characteristics of a test cell in order to more appropriately select a treatment method for a malignant tumor.
A genetic marker for evaluating cell characteristics of a test cell is provided. Genetic marker, which is a polynucleotide comprising mRNA, peptide or protein of a nicotinamide phosphoribosyltransferase (NAMPT) gene, or a NAMPT gene promoter sequence. The NAMPT gene is an indicator gene that is highly expressed in tumors with high metastasis and is highly correlated with tumor cell metastasis, and is used to evaluate the cell characteristics of the subject's tumor cells. Genetic markers that can be used.
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Description

  Embodiments described herein relate generally to a genetic marker, a method, a reporter vector, a primer set and an assay kit, and a cell property evaluation apparatus for evaluating the cell property of a test cell.

  Cancer is the number one cause of death in Japan. Cancer is a group of diseases having a common malignant tumor formation, and more than 200 various tumors have been identified to date. A common feature of malignant tumors is the uncontrolled proliferation of cells, the ability to invade nearby tissues, and the ability to form metastases to systemic tissues.

  In the treatment of cancer (malignant tumor), it is important to select an appropriate treatment method according to the nature of the tumor (characteristics of the tumor cells). In order to select an appropriate treatment, it is desirable to evaluate the characteristics of tumor cells, that is, the benign malignancy of the tumor, as early as possible. In the case of a malignant tumor, it is desirable to be able to evaluate its aggressiveness, that is, invasiveness and metastasis. However, at present, it is difficult to evaluate the metastasis from the primary tumor, that is, the tumor cell obtained from the lesion where the tumor first occurred.

  Tumor diagnosis includes diagnosis using an image diagnostic apparatus such as MRI and CT, and diagnosis performed by observing cells collected from the tumor. The cell characteristics include morphological characteristics, that is, characteristics that can be distinguished from the appearance such as the size and shape of the cells and adhesiveness. Known methods for observing the morphological characteristics of cells include Papanicolaou staining and Giemsa staining. In such a staining method, cell nuclei and cytoplasm are dyed separately, and the characteristics of tumor cells are evaluated from their morphological characteristics.

  On the other hand, there are also cell characteristics that cannot be distinguished from the morphology. Such cellular properties are accompanied by, for example, changes in gene expression. In gene expression, a specific protein binds to a 5 'upstream promoter sequence of a gene to activate the promoter. The activated promoter promotes gene transcription to synthesize mRNA, and the mRNA is translated to synthesize proteins. Changes in gene expression are detected by examining the promoter activity of the gene, the amount of mRNA, and the amount of protein.

  For example, when a change in expression of a specific gene is highly correlated with the characteristics of tumor cells, the gene can be used as a tumor marker. By detecting a tumor marker, it is possible to discriminate cell characteristics related to gene expression. For example, if a specific tumor marker is detected in blood, another body fluid, or collected tumor cells, the presence of tumor cells highly correlated with the marker is suggested.

  Known tumor markers include the Ki-67 gene that evaluates the proliferation of tumor cells. This tumor marker is used for evaluating the proliferation of tumor cells. In addition, Arf-6, Glut1 and the like have been reported as tumor marker candidates for evaluating tumor cell invasion and metastatic properties. However, the accuracy of the evaluation is not sufficient.

  In such a situation, in order to be able to select a malignant tumor treatment more appropriately, development of a further technique for evaluating cell characteristics is desired.

Special table 2011-508609 gazette Special table 2012-533530 gazette International Publication No. 2004/058990

Cancer Biology & Therapy, May 2008, Vol. 7, No. 5, p663-670 Endocrinology, February 2012, Volume 153, No. 2, p554-563

  Embodiment aims at providing the means for evaluating the cell characteristic of a test cell.

  According to the embodiment, a genetic marker for evaluating the cell characteristics of a test cell is provided. The gene marker is a polynucleotide containing the mRNA, peptide or protein of the NAMPT gene, or the NAMPT gene promoter sequence.

The schematic diagram which shows one example of embodiment. The schematic diagram which shows one example of embodiment. The block diagram which shows an example of embodiment. The block diagram which shows an example of embodiment. The flowchart which shows an example of embodiment. The figure which shows an experimental result. The figure which shows an experimental result. The figure which shows an experimental result. The schematic diagram which shows the structure of the vector which is an example of embodiment used for experiment. The figure which shows an experimental result. The figure which shows an experimental result. The figure which shows an experimental result.

  Hereinafter, embodiments will be described with reference to the drawings.

  The embodiment has discovered that the NAMPT gene is highly expressed in highly metastatic tumor cell lines, revealed that it is an indicator gene highly correlated with the metastasis of tumor cells, and the cell characteristics of test cells Based on the finding that it can be used as a genetic marker for evaluating

  The NAMPT gene is a gene encoding nicotinamide phosphoribosyltransferase, and is a rate-limiting enzyme in mammalian NAD biosynthesis. The NAMPT gene is highly expressed in highly metastatic tumor cell lines.

  On the other hand, the metastasis of cells is one of the important indexes for evaluating the cell characteristics of test cells. Therefore, by measuring the expression level of the NAMPT gene, it is possible to evaluate the cell characteristics of the test cell, for example, the presence or absence of metastatic or invasiveness, whether the cell is normal or abnormal It is. If the test cell is abnormal or possibly abnormal, the degree of abnormality or malignancy can be evaluated. Therefore. The genetic marker based on the NAMPT gene makes it possible to evaluate the cell characteristics of tumor cells more accurately than before.

  The evaluation of the cell characteristics of the test cell is to evaluate the cell characteristics of the test cell, for example, the presence or absence of metastasis or invasiveness, whether the cell is normal or abnormal, and the test cell Is abnormal or possibly, includes evaluating the degree of abnormality or the degree of malignancy. For example, when the test cell is a tumor cell, the evaluation of the cell characteristic is, for example, the evaluation of the cell characteristic of the tumor cell, for example, whether the tumor cell is metastatic or invasive, the tumor cell metastasis It may be to evaluate the degree of sex, the degree of benign or malignant tumor cells, the degree of normality or abnormality of tumor cells.

  Here, when describing as “tumor cells”, they may be understood as “cancer cells” or collectively “cancer cells”.

  The test cell to be examined may be a cell whose cell characteristics are to be evaluated.

  Such a method for evaluating the cell characteristics of the test cell may be characterized by using the expression level of the NAMPT gene in the test cell as an index. For example, in this method, using the expression level of the NAMPT gene as an indicator includes measuring the expression level of the NAMPT gene in the test cell, comparing the measured expression level of the test cell with a threshold, Evaluation of the cell characteristics of the test cell based on the result of the comparison and based on the determination may be included.

  This evaluation may be performed in comparison with the expression level or degree of expression of the NAMPT gene in the reference cell. Alternatively, this evaluation can be performed in consideration of the cell characteristic of the reference cell in addition to the expression level of the NAMPT gene in the reference cell.

  Examples of the reference cell may be a healthy cell or a normal cell, a tumor cell whose cell characteristics such as the presence or absence of metastasis and / or the degree of metastasis have been clarified. The expression level or cell characteristics of the NAMPT gene in the reference cell, for example, the degree of metastasis, may be measured in advance, and may be measured and evaluated simultaneously when performing a test using the gene marker of the embodiment. . In addition, the expression level or metastatic level of the reference gene obtained from the reference cell may be set as a threshold value. Such a threshold value may be a predetermined threshold value, and the cell characteristics of the test cell may be determined. It may be measured at the same time as the evaluation, or may be determined before and after evaluating the cell characteristics of the test cell.

  The gene marker for evaluating the cell characteristics of the test cell may be any substance that can be used for measuring the presence and / or expression level of the NAMPT gene. For example, the genetic marker may be a NAMPT gene mRNA, peptide or protein, or a polynucleotide comprising a NAMPT gene promoter sequence.

Examples of NAMPT gene promoter sequences are shown in Table 1-1 and Table 1-2.

  This is the sequence represented by SEQ ID NO: 1, and is an example of a human-derived NAMPT gene promoter sequence. However, the NAMPT gene promoter sequence is not limited to this sequence, and may contain one to several mutations such as substitution, deletion, and addition as long as the NAMPT gene promoter sequence activity is exhibited.

  A further embodiment is a method for evaluating cell characteristics of test cells using such genetic markers. In this method, for example, the cellular characteristics of the test cell may be evaluated using the expression level of the NAMPT gene mRNA, the expression level of the peptide or protein, or the activity of the NAMPT gene promoter (NAMPT promoter activity) as an index. According to the method of the embodiment, it is possible to evaluate the cell characteristics of the test cell more accurately at an earlier stage. In the conventional method, it is difficult to evaluate the metastasis from the primary tumor, that is, the tumor cell of the lesion in which the tumor first occurred. However, according to the embodiment, for example, the tumor cell from the primary tumor is also evaluated. It becomes possible to evaluate cell characteristics.

  A further embodiment provides a method for evaluating tumor cell characteristics using NAMPT gene expression as an indicator. Specifically, the expression of the NAMPT gene in the test cells collected from the tumor of the subject may be quantitatively detected, and the expression of the gene may be compared with the expression of the NAMPT gene in healthy cells as controls. When the expression level of the NAMPT gene in the test cell is higher than the NAMPT gene in the healthy cell, it can be determined that the test cell is highly metastatic or abnormal. When the expression level of the NAMPT gene in the test cell is lower or equivalent to the NAMPT gene in the healthy cell, it can be determined that the test cell has low metastasis or low abnormality.

  The test cell may be a cell group or a single cell. The test cells may be collected from the subject's tumor by a known collection method. In the present invention, tumor cells collected from epithelial tumors are preferred. Among epithelial tumors, tumor cells collected from breast tumors and lung tumors are more preferred as test cells. However, it is not limited to these. For example, blood, urine, feces, saliva, tissue obtained by biopsy, oral mucosa, cells contained in body cavity fluid or sputum, or cultured cells may be used as test cells.

  In the cell characteristic evaluation of the test cell according to the embodiment, the expression level of the NAMPT gene in the test cell may be compared with the expression of the NAMPT gene in a healthy cell. An average expression level of the NAMPT gene in healthy cells may be detected in advance, a threshold value in cell characteristic evaluation may be set from this value, and this threshold value may be compared with the NAMPT expression level in the test cell. Therefore, it is not always necessary to detect the expression level of the NAMPT gene in healthy cells.

  The expression of the NAMPT gene may be performed by detecting the activity of the NAMPT protein, NAMPT mRNA, or NAMPT gene promoter. These may be used as an index of the gene expression or the expression level of the gene.

  When NAMPT protein is detected, it may be detected quantitatively by enzyme immunization (EIA) or fluorescence immunization (FIA). Alternatively, detection may be performed using an immune cell staining method (ICC), an immunohistochemical staining method (IHC), or an automatic cell analysis / separation apparatus (FACS).

  For the expression of the NAMPT gene, for example, NAMPT mRNA may be detected. In that case, NAMPT mRNA may be detected by any method. For example, using a primer set, a sample may be amplified using a PCR amplification method, a LAMP amplification method, a SMAP amplification method, an ICAN amplification method, etc., and the amplification product may be analyzed. In this case, a reverse transcription PCR method, a reverse transcription LAMP amplification method, a reverse transcription SMAP amplification method, a reverse transcription ICAN amplification method, or the like that directly amplifies mRNA may be preferably used. Analysis of the amplification product may be performed by detecting the binding with the probe nucleic acid, or the base sequence of the amplification product may be directly specified. Alternatively, detection may be performed by determining the base sequence of a nucleic acid that is a NAMPT gene expression product of a test cell without amplification.

Examples of preferred primer sets are shown in Table 2.

  The primer sets shown in Table 2 are primer sets for reverse transcription PCR amplification. SEQ ID NO: 2 and SEQ ID NO: 3 are used in combination. The primer of SEQ ID NO: 2 is a forward primer, and the primer of SEQ ID NO: 3 is a reverse primer. These primer sets are preferably used for amplifying and detecting NAMPT mRNA, but the primer set for amplifying and detecting NAMPT mRNA is not limited thereto.

  When detecting the activity of the NAMPT gene promoter, it may be detected by a method using a reporter vector incorporating the promoter of the NAMPT gene. The reporter vector may be a normal reporter vector, that is, a non-replicating (i.e., non-amplifying) reporter vector in a mammalian cell, or a self-amplifying reporter vector, i.e., autonomously replicating in a mammalian cell (i.e., A reporter vector that amplifies) may be used.

  Hereinafter, a method for evaluating the cell characteristics of the test cells will be described in more detail.

1. 1. Cell characterization of test cells by detection of NAMPT protein 1-1. Detection Method and Cell Characteristic Evaluation Method The NAMPT protein may be detected using an antibody specific to the NAMPT protein (ie, an anti-NAMPT antibody). Such an antibody may be newly prepared by using the full length or a part of the NAMPT protein as an antigen, or a commercially available antibody may be used.

  Examples of commercially available antibodies include, but are not limited to, rabbit anti-NAMPT polyclonal antibodies (ab45890, Abcam). What is necessary is just to produce in accordance with the well-known antibody production method, when producing a new antibody.

  The detection of the NAMPT protein using the anti-NAMPT antibody may be performed by a known protein detection method. Such protein detection methods include, for example, enzyme immunization (EIA), fluorescence immunization (FIA), immune cell staining (ICC), immunohistochemical staining (IHC), or automatic cell analysis separation device (FACS). However, it is not limited to these.

  In EIA or FIA, for example, a protein extracted from a test cell collected from a tumor and a protein extracted from a healthy cell as a control are each immobilized in a well of a microtiter plate. Thereafter, an anti-NAMPT antibody is added and reacted specifically with the NAMPT protein, and then excess antibody is removed by washing with a phosphate buffer or the like. Thereafter, detection is performed using a labeled antibody that recognizes the anti-NAMPT antibody.

  As the labeled antibody, an enzyme-labeled antibody or an antibody labeled with a fluorescent dye may be used. When an enzyme-labeled antibody is used, after reacting the anti-NAMPT antibody with the labeled antibody, the excess labeled antibody is removed by washing with a phosphate buffer or the like. Then, after adding an enzyme substrate and carrying out an enzyme reaction, the reaction product of the enzyme reaction may be detected. For example, when the enzyme of the labeled antibody produces a luminescent substance from the substrate, the luminescence intensity of the well may be measured with a luminometer. Thereby, it is possible to detect the amount of luminescent substance quantitatively. Under appropriate conditions, the luminescence intensity correlates with the amount of labeled antibody, the amount of labeled antibody correlates with the amount of anti-NAMPT antibody, and the amount of anti-NAMPT antibody correlates with the amount of NAMPT protein. Thereby, the amount of NAMPT protein in the protein extracted from the cells can be quantified by quantitatively measuring the amount of the luminescent substance. Similarly, when a fluorescent substance is produced from the substrate of the labeled antibody enzyme, the amount of NAMPT protein can be quantified by measuring the fluorescence intensity of the well with a fluorometer. In detection by fluorescence intensity, the labeled antibody may be directly labeled with a fluorescent substance. When the enzyme of the labeled antibody produces a chromogenic substance from the substrate, the amount of NAMPT protein can be quantified by measuring the absorbance of the well with an absorptiometer. EIA or FIA detects the amount of NAMPT protein in test cells and healthy cells and compares the amount of protein in both cells, and the expression level of NAMPT gene in the test cells is higher than the expression level of NAMPT gene in healthy cells In this case, it may be determined that the abnormality of the test cell is high, for example, the metastasis of the test cell is high. However, the measurement of the expression level of the NAMPT protein in healthy cells is not necessarily performed together with the measurement of the expression level of the NAMPT gene in the test cell. For example, an average NAMPT protein amount of healthy cells may be measured in advance, and this value may be set as a threshold value in cell characteristic evaluation. Cell characteristics may be evaluated by comparing this threshold with the amount of NAMPT protein in the test cell.

  For example, in ICC and IHC, tumor cells (tissue) as test cells collected from a tumor and healthy cells (tissue) as a control are immobilized with an immobilization solution, and then unreacted with a phosphate buffer or the like. The immobilizing solution can be removed to perform a blocking treatment. After adding an anti-NAMPT antibody to react specifically with the NAMPT protein, excess antibody can be removed by washing with a phosphate buffer or the like, and detection can be performed using a labeled antibody that recognizes the anti-NAMPT antibody. Reagents used for ICC and IHC may be appropriately selected from known reagents and used. As the labeled antibody, an enzyme-labeled antibody or an antibody labeled with a fluorescent dye can be used. In ICC and IHC, the expression of NAMPT protein can be detected with a light microscope. For example, when a luminescent substance is produced from the substrate by the enzyme of the labeled antibody, the luminescence of the cell can be detected by a luminescence microscope. When the fluorescent substance is generated from the substrate of the enzyme of the labeled antibody, the fluorescence of the cell can be detected by a fluorescence microscope. When the enzyme of the labeled antibody produces a color developing substance from the substrate, the color development of the cells can be detected with an optical microscope. When detecting by fluorescence, the labeled antibody may be directly labeled with a fluorescent substance. In ICC or IHC, the intensity of staining, that is, the intensity of luminescence, fluorescence or color development, may be observed or compared with a microscope as the expression level of NAMPT protein in test cells and healthy cells. Thereby, for example, when the expression level (staining intensity) of the NAMPT gene in the test cell is higher than the expression level (staining intensity) of the NAMPT gene in the healthy cell, for example, there is an abnormality in the test cell. It can be determined that there is a possibility that the test cell is highly abnormal, or that the test cell is highly metastatic.

  In FACS, after immobilizing tumor cells as test cells collected from a tumor and healthy control cells with an immobilization solution, the unreacted immobilization solution is removed with a phosphate buffer or the like to perform blocking treatment. Can be broken. After adding an anti-NAMPT antibody and reacting specifically with the NAMPT protein, the excess antibody is washed away with a phosphate buffer or the like, and the protein is detected using a labeled antibody that recognizes the anti-NAMPT antibody. obtain. In FACS, the labeled antibody is preferably an antibody labeled with a fluorescent substance. Cells prepared in this way can be detected by FACS. A commercially available apparatus such as FACS Verse sold by BD (Becton, Dickinson and Company) can be used for FACS. In FACS, as the expression level of NAMPT protein in test cells and healthy cells, the fluorescence intensity of the cells is detected and compared, and the expression level (fluorescence intensity) of NAMPT gene in the test cells is the expression level of NAMPT gene in healthy cells. When the amount is higher than the amount (fluorescence intensity), for example, it can be determined that there is an abnormality or the metastatic property of the test cell is high.

1-2. Kit for evaluating cell characteristics of test cells by detection of NAMPT protein In the present invention, a kit for cell specific evaluation of test cells by detection of NAMPT protein is provided as a further embodiment. For example, it may be a kit for evaluating metastasis of tumor cells by detecting NAMPT protein. The cell characterization kit includes at least one anti-NAMPT antibody and a buffer. The cell characterization kit may include a container that contains a known carrier carrying an anti-NAMPT antibody and / or a reporter vector. Furthermore, any reagent necessary for performing the method for evaluating the cell characteristics of the test cells may be included. For example, it may further contain a labeled antibody that recognizes the anti-NAMPT antibody, a substrate for detecting the labeled antibody, an excipient, a stabilizer, and / or a component having other desired effects. The kit composition may be provided in a container.

2. 2. Cell characteristic evaluation of test cells by detection of NAMPT mRNA 2-1. Detection Method and Cell Characteristic Evaluation Method NAMPT mRNA may be detected by reverse transcription PCR using a primer set specific for NAMPT mRNA. However, the method is not limited to this method, and a known mRNA detection method may be used as long as the mRNA can be detected. Examples of such a detection method include Northern blotting using a probe that specifically detects NAMPT mRNA, DNA / RNA chip method, and the like.

  In the reverse transcription PCR method, for example, cDNA is synthesized by reverse transcription reaction using RNA extracted from test cells collected from a tumor and healthy control cells as a template, and a primer set specific to the template is used. A part or the full length of NAMPT mRNA can be amplified and detected by the PCR used.

  The reverse transcription PCR method is a method for detecting mRNA that combines the above reverse transcription reaction and PCR. Commercially available reagents such as enzymes, substrates and buffers used for reverse transcription and / or PCR can be used. Alternatively, a kit in which the reagent is packaged in advance may be used.

  The primer set used for reverse transcription PCR can include a combination of primers each having a complementary nucleotide sequence to the sense strand and the antisense strand encoding the NAMPT protein. The primer set is preferably a combination of primers spanning two or more exons so as not to amplify genomic DNA mixed in RNA. Examples of such primer sets can be the primer sets set forth in SEQ ID NO: 2 and SEQ ID NO: 3. However, the present invention is not limited to this, and the primer set only needs to specifically amplify a part or the entire length of NAMPT mRNA. The amount of NAMPT mRNA contained in RNA extracted from cells may be quantified by amplifying a part or full length of NAMPT mRNA by reverse transcription PCR using the above primer set under appropriate reaction conditions.

  For example, when the amount of NAMPT mRNA in a test cell and a healthy cell is detected by reverse transcription PCR and the amount of mRNA in both cells is compared, and the amount of NAMPT mRNA in the test cell is higher than the expression level of NAMPT mRNA in the healthy cell The test cell may be abnormal, for example, it may be determined that the test cell is highly metastatic. At this time, the measurement of the amount of NAMPT mRNA in healthy cells is not necessarily performed simultaneously with the measurement of the amount of NAMPT mRNA in test cells. The average amount of NAMPT mRNA in healthy cells may be measured in advance, and this value may be set as a threshold value for cell characteristic evaluation. Cell characteristics can be evaluated by comparing such a threshold value with the amount of NAMPT mRNA in the test cell.

2-2. Kit for evaluating cell characteristics of test cells by detecting NAMPT mRNA As a further embodiment, a kit for evaluating cell characteristics of test cells by detecting NAMPT mRNA is provided. It can be, for example, a cell characterization kit for tumor cells by NAMPT mRNA detection. The cell property evaluation kit may include the primer set and the buffer described in 1-1. The cell characterization kit may include a known carrier carrying a primer set and / or a container containing the primer set. Further, the cell property evaluation kit may include any reagent necessary for performing a method for evaluating the cell property of a test cell. For example, an enzyme, a substrate, a buffer solution, an excipient, a stabilizer, and / or a component having other desired effects for performing reverse transcription PCR may be included. The kit composition may be provided in a container.

3. 3. Detection of NAMPT gene promoter activity using reporter vector 3-1. Reporter Vector An example of a reporter vector that can be used for detection of NAMPT gene promoter activity is described with reference to FIG. The reporter vector shown in FIG. 1 is an example of a reporter vector that does not autonomously replicate (ie, amplify) in mammalian cells. The reporter vector 1 includes a reporter gene expression unit 2 that produces a detectable signal. The reporter gene expression unit 2 includes a NAMPT promoter sequence 4, a reporter gene 5 operably linked downstream thereof, and a transcription termination signal sequence 6 operably linked downstream thereof.

  Here, “functionally linked” refers to a state in which each gene is linked in a state where it can exert its intended function. For example, in the case of a sequence encoding a protein, it means that the amino acid sequences encoded by the respective base sequences are correctly linked. In other words, it means that there is no shift in the frame of the amino acid codon and a peptide that functions in the target activity is synthesized in the cell into which the base sequence is introduced. Also, herein, the term “functionally coupled” may be used interchangeably with the term “operably coupled”.

  The promoter sequence 4 of the NAMPT gene is a nucleic acid sequence from which information for controlling the transcription activity from the NAMPT gene to mRNA should be obtained as the promoter activity of the gene. The promoter sequence 4 of the NAMPT gene is a sequence located in the 5 'upstream region of the nucleic acid sequence encoding the amino acid sequence information of the NAMPT gene, that is, the NAMPT protein, in the cell genome. The promoter sequence 4 of the NAMPT gene may be a nucleic acid sequence having a length sufficient to obtain information for controlling transcription activity from the NAMPT gene to mRNA. For example, an example of promoter sequence 4 of NAMPT gene is described in SEQ ID NO: 1. However, the promoter sequence of the gene need not completely match the nucleotide sequence described in SEQ ID NO: 1 as long as information for controlling the transcription activity from the NAMPT gene to mRNA can be obtained. The base sequence may be a base sequence shorter than SEQ ID NO: 1, that is, only a part of the sequence described in SEQ ID NO: 1. Further, it may be a fusion nucleic acid sequence obtained by linking a part of the nucleic acid sequence shown in SEQ ID NO: 1 and the nucleic acid sequence of another gene promoter. For example, the promoter sequence of the NAMPT gene may be obtained from the human genome using a primer set including a forward primer and a reverse primer respectively including SEQ ID NO: 4 and SEQ ID NO: 5 shown in Table 2.

  In the above description, “sequence having homology” means a sequence that is identical to a specific sequence on the genome, or a part of the same sequence as the specific sequence on the genome, for example, one or several sequences. A sequence in which a base is substituted, deleted or added, and has a promoter activity. For example, “having homology”, “homologous”, and “homologous” may be sequences that are 90% or more homologous to the genome sequence. The “fusion nucleic acid sequence” refers to a sequence obtained by integrating two or more specific sequences on different genomes or on the same genome. That is, the promoter sequence 4 of the NAMPT gene may be composed of a part of the NAMPT gene promoter sequence and two or more units composed of other gene promoter sequences. The promoter sequence 4 of the NAMPT gene may include any further sequence in addition to the sequence having homology to the sequence on the genome of the test cell. When any additional sequence is included, any additional sequence called a linker sequence or the like may be included as long as the promoter sequence 4 of the NAMPT gene has promoter activity.

  Reporter gene 5 may be any gene that encodes a protein that produces a detectable signal. The detectable signal may be any detectable signal generated from the reporter protein itself, the reaction between the reporter protein and any substance, the binding between the reporter protein and any substance, or the like.

  The reporter gene 5 encoding the reporter protein may be any reporter protein that produces a detectable signal. The reporter gene 5 may be, for example, a luciferase gene, β-galactosidase gene, nitric oxide synthase gene, xanthine oxidase gene, blue fluorescent protein gene, green fluorescent protein gene, red fluorescent protein gene, and heavy metal binding protein gene. . Each of these genes encodes luciferase, β-galactosidase, nitric oxide synthase, xanthine oxidase, blue fluorescent protein, green fluorescent protein, red fluorescent protein and heavy metal binding protein, and these proteins are activated by activation of the reporter vector. To express. A preferable example in one embodiment is a gene encoding a luminescent enzyme protein, and includes, for example, a luciferase gene and a β-galactosidase gene.

  An example of a reporter vector that expresses a heavy metal binding protein as a detectable signal is disclosed in Japanese Patent Application Laid-Open No. 2012-200245.

  The signal obtained from the reporter gene may be any signal that can be detected by any device. Further, such signal detection may be performed using any device selected according to the type of signal. Examples of the apparatus may be a luminescence measuring apparatus, a fluorescence measuring apparatus, an X-ray measuring apparatus, an electron spin resonance apparatus, a nuclear medicine diagnostic apparatus, a magnetic resonance imaging apparatus, and the like. Further, the result obtained by detecting the signal may indicate the presence or absence of the signal, the magnitude of the signal, or both.

  Preferred examples of detectable signals are optical signals such as luminescence, fluorescence and color development. As used herein, “luminescence” refers to chemiluminescence, bioluminescence or biochemiluminescence. When the signal is an optical signal, the signal may be detected by an optical detection device. For example, when the detectable signal is light emission, the signal may be detected by a light emission measuring device capable of detecting light emission, for example, a light emission microscope.

  The transcription termination signal sequence 6 may be a sequence that terminates transcription of a gene upstream thereof, and may be, for example, a poly (A) addition signal. The poly (A) addition signal may be any signal that functions to terminate transcription of mammalian genes. Examples include the late poly (A) addition signal (SEQ ID NO: 6) of the SV40 virus and the poly (A) addition signal (SEQ ID NO: 7) of the bovine growth hormone gene. Examples of transcription termination signal sequences are shown in Table 3.

The transcription termination signal shown in Table 3 may be linked upstream of the promoter sequence 4 of the NAMPT gene. By linking the transcription termination signal 6 upstream of the promoter sequence 4, expression (leakage) of the reporter gene 5 due to inappropriate transcription from any sequence of the reporter vector can be prevented. Thereby, the background (noise) due to inappropriate transcription (expression leak) can be reduced, and the detection sensitivity and detection accuracy of the signal generated by the expression of the reporter gene 5 can be increased.

  However, the poly (A) addition signal that can be used in the practice of this embodiment is not limited to this, and the modified poly (A) addition signal may be used as long as the function as the poly (A) addition signal is not impaired. It may be used.

  An example of a self-amplifying reporter vector will be described with reference to FIG. 2A and 2B show two examples of self-replicating vectors.

  The self-replicating vector 21 shown in FIG. 2 (a) has the same configuration as the reporter vector shown in FIG. 1 except that it contains a replication initiation sequence.

  The self-replicating vector 21 includes a reporter gene expression unit 2 and a replication initiation unit linked downstream thereof. The example illustrated in FIG. 2 is an example in which the replication start unit includes a replication start sequence 3. The replication initiation sequence 3 initiates self-replication in cells into which the self-replication vector 21 has been introduced by binding of the corresponding replication initiation protein.

The self-replicating vector performs self-replication in the test cell nucleus under the condition that the replication initiation protein is expressed in the test cell. The replication initiation sequence 3 may be a sequence that is activated by binding of a replication initiation protein and initiates self-replication in mammalian cells, for example, and may be a replication initiation sequence known per se. The replication initiation sequence 3 may be, for example, a replication initiation sequence derived from simian virus 40 (SEQ ID NO: 8), Epstein-Barr virus, or mouse polyoma virus. An example of the replication start sequence 3 is shown in Table 4.

  As the self-replicating vector, for example, a plasmid vector known per se may be used. For example, an example of a plasmid vector of this self-replicating vector is disclosed in JP2013-42721.

  Self-replication of the self-replicating vector is initiated by binding of a replication initiator protein to the replication initiator sequence 3. The replication initiator protein may be present together with the self-replicating vector in the test cell nucleus so that it can bind to the replication initiator sequence 3. For example, the replication initiator protein may be expressed from a self-replicating vector used as a reporter vector, may be expressed from a further vector, or may be expressed by a test cell.

  For example, a replication initiator protein unit may be present in order to supply a replication initiator protein into the test cell nucleus. The replication initiation protein unit may include, for example, a promoter, a replication initiation protein gene encoding a replication initiation protein operably linked downstream thereof, and a transcription termination signal sequence operably linked downstream thereof. Alternatively, the replication initiator protein unit may consist of these sequences.

  The replication initiating protein unit may be present on the nucleic acid of the self-replicating vector 21 or may be present on a nucleic acid different from the nucleic acid of the self-replicating vector 21. When the replication initiator protein unit is present on a nucleic acid different from that of the self-replicating vector, the replication initiator protein unit may be present on the chromosome of the test cell or may be present on another nucleic acid contained in the test cell. May be.

  When the replication initiating protein unit is contained in a self-replicating vector as a reporter vector, the expression of the reporter gene expression unit 2 and the replication initiating protein unit contained therein are both controlled by one promoter sequence 4. Alternatively, their expression may be controlled by two independent promoter sequences. When their expression is controlled by the two promoter sequences, for example, the replication initiator protein unit may be present on the nucleic acid of the self-replicating vector 21 independently of the reporter gene expression unit 2. In that case, the promoter controlling the expression of the replication initiator protein gene is preferably a constitutively expressed promoter.

The replication initiation protein gene may be, for example, the large T antigen gene of Simian virus 40, the EBNA-1 gene of Epstein-Barr virus, the large T antigen gene of mouse polyoma virus, and the like. Examples of preferred large T antigen genes include a nucleic acid represented by SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11, or a deletion of one or several bases in the sequence of SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11 Or a nucleic acid having a DNA replication initiation function, which is indicated by the substituted or added sequence. Here, the DNA replication initiation function refers to encoding a protein capable of initiating replication. Examples of the replication initiation protein gene are shown in Table 5-1, Table 5-2, Table 6-1, Table 6-2, Table 7-1 and Table 7-2.

The combination of the replication initiation protein gene and the replication initiation sequence may be any combination that can initiate replication. Examples of such combinations are:
A combination wherein the gene encoding the replication initiator protein is a large T antigen gene of simian virus 40 and the replication initiator sequence is a replication initiator sequence from simian virus 40;
A combination in which the replication initiator protein gene is the Epstein-Barr virus EBNA-1 gene and the replication initiator sequence is the replication initiator sequence from Epstein-Barr virus;
A combination wherein the replication initiator protein gene is a large T antigen gene of mouse polyoma virus and the replication initiator sequence is a replication initiator sequence from mouse polyoma virus. Combinations such as these are preferred, but are not limited thereto.

The self-replicating vector may be a bicistronic expression vector. In the bicistronic expression vector, by using the bicistronic expression sequence, the NAMPT promoter sequence 4 simultaneously controls the expression of two genes included downstream thereof, that is, the reporter gene and the replication initiation protein. The That is, in such a self-replicating vector, the reporter gene expression unit 2 and the replication initiating protein unit are expressed by being controlled by the same promoter. An example of such a bicistronic expression self-replicating vector will be described with reference to FIG. The self-replicating vector 31 includes a NAMPT promoter sequence 4, a reporter gene 5, an IRES 7, a replication initiation protein gene 8, a transcription termination signal sequence 6, and a replication initiation sequence 3. The NAMPT promoter sequence 4, the reporter gene 5, the IRES 7, the replication initiation protein gene 8, and the transcription termination signal sequence 6 are functionally linked in this order from upstream to downstream. IRES7 is an internal ribosomal entry site and is an example of a bicistronic expression sequence. An example of a bicistronic expression sequence is, for example, a brain myocardial virus internal ribosome entry sequence (SEQ ID NO: 12). IRES7 is a base sequence that initiates protein translation by binding a ribosome to the interior of mRNA without depending on the 5 ′ end cap structure of mRNA in mammalian cells. By placing IRES7 between the two genes, bicistronic mRNA is synthesized. That is, by incorporating IRES7 between two genes, two types of proteins encoded by these two genes can be translated into one mRNA. This example is shown in Table 8.

  Due to the presence of IRES7, the sequence of the reporter gene 5 is transcribed by the NAMPT promoter sequence 4, and subsequently, the replication initiation protein gene 8 is transcribed. After the sequence of the replication initiation protein gene 8 is read, the transcription is stopped by the presence of the transcription termination signal sequence 6. The replication initiator protein transcribed and translated from the replication initiator protein gene 8 is bound to the replication initiator sequence 3, and the replication of the self-replicating vector 31 is started.

  By detecting a detectable signal generated from such a reporter vector, it is possible to evaluate the activation of the NAMPT promoter sequence, and thereby the expression level of the NAMP gene can be evaluated. Based on the result, the cell characteristics of the test cell can be determined.

  In the above reporter vector, promoter activity means inducing transcription of a gene functionally connected downstream of the promoter sequence. When the promoter sequence 4 of the NAMPT gene has promoter activity, the promoter activity is derived from the partial sequence of the promoter sequence 4 of the NAMPT gene, even if it is derived from the entire sequence of the promoter sequence 4 of the NAMPT gene It may be. For example, the promoter sequence 4 of the NAMPT gene may contain a minimal promoter. Promoter activation of the promoter sequence 4 of the NAMPT gene is controlled by the presence or absence of binding of a transcription factor to a partial sequence on the promoter sequence 4 of the NAMPT gene and the modification state of the base to be modified.

  The promoter sequence 4 of the NAMPT gene includes a target nucleic acid sequence that is a sequence having identity with a portion on the genome of the test cell. Therefore, in the case of a reporter vector that self-amplifies, the self-replicating vector contains the target nucleic acid sequence in the target nucleic acid sequence with the state of substitution of the functional group in the target nucleic acid sequence contained in the test cell as an epigenic characteristic during self-amplification. It can be copied (ie, copied) onto the base. That is, the activation of the NAMPT promoter sequence may be activated or deactivated depending on the presence or absence or degree of substitution of a functional group in a modifiable base in the target nucleic acid sequence, for example, a methyl group or an acetyl group. May be.

  The self-replicating reporter vector 31 can self-replicate in the test cell under the condition that the NAMPT gene promoter 4 is activated. That is, when the NAMPT promoter 4 is activated, the reporter gene 5 and the replication initiation protein gene 8 are transcribed as bicistronic mRNA by the action of IRES7, and the reporter protein and the replication initiation protein which are translation products of the mRNA are produced. Is done. The replication initiator protein binds to the replication initiation sequence 3 of the self-replicating reporter vector, and binds to a protein necessary for DNA replication, so that the reporter vector self-replicates.

  The self-replicating reporter vector may be a vector capable of self-replication, and may be, for example, a plasmid vector. Examples of such self-replicating plasmid vectors are disclosed in JP2013-42721.

3-2. Detection of NAMPT Promoter Activity An example of a method for detecting the promoter activity of the NAMPR gene (ie, NAMPT promoter activity) in test cells or healthy cells using the reporter vector described in 3-1. Here, the detection of promoter activity may be detection of the presence or absence of promoter activity or quantitative measurement of promoter activation.

  A reporter vector 1 having a promoter region of the NAMPT gene (SEQ ID NO: 1) or a self-amplifying reporter vector 21 or 31 is prepared (a). Next, the reporter vector 1, 21, or 31 is introduced into living test cells and healthy cells (b). Thereafter, the state in which the reporter gene of the reporter vector 1, 21, or 31 is expressed in the cell is maintained. When the reporter vector 21 or 31 is used, it is maintained in a culture condition in which the cells can divide and grow, that is, in a state where the reporter vector can self-replicate (c). In such a state, the promoter activity of the NAMPT gene of the cell genome is reflected in the activity of the NAMPT promoter (SEQ ID NO: 1) of the reporter vector (d). Next, the amount of reporter protein expressed from the reporter gene is measured as an index of NAMPT promoter activity (e). Finally, when the amount of the reporter protein in the test cell and the healthy cell is compared, and the amount of the reporter protein in the test cell is large, the test cell is an abnormal cell, for example, a tumor cell Determine (f). At this time, NAMPT promoter activity in healthy cells, that is, introduction of a reporter vector into healthy cells and measurement of reporter protein expression level are not necessarily required. For example, the average NAMPT promoter activity of healthy cells is measured in advance, this value is set as a threshold value for cell characteristic evaluation, and the evaluation is performed by comparing this threshold value with the NAMPT promoter activity of the test cell. May be.

  The NAMPT promoter activity described above can be detected by detecting or quantifying the expression of the reporter protein. The reporter protein may be, for example, luciferase, β-galactosidase, nitric oxide synthase, xanthine oxidase, blue fluorescent protein, green fluorescent protein, red fluorescent protein, and heavy metal binding protein. An appropriate detection and / or measurement method can be selected according to the type of the selected reporter protein.

  Examples of reporter protein detection are given below. When the reporter gene is a luminescent gene, for example, a luciferase gene, the luminescence intensity of the solution can be measured using a luminometer after extracting the luciferase protein from the test cell and adding the substrate. When the reporter gene is a fluorescent protein gene, for example, the test cell can be irradiated with light of a specific wavelength, and the intensity of the fluorescence generated from the fluorescent protein in the test cell can be measured by a fluorometer. Alternatively, the extract containing the fluorescent protein extracted from the test cells can be irradiated with light of a specific wavelength, and the intensity of the fluorescence generated from the fluorescent protein in the extract can be measured by a fluorescence measuring device.

  When the reporter gene is a β-galactosidase gene, the absorbance of the solution can be measured using an absorbance measurement device after extracting the β-galactosidase protein and adding a substrate.

  As the reporter gene, an active oxygen generating enzyme gene may be used. The active oxygen generating enzyme gene is a gene encoding an enzyme that generates active oxygen. Examples of the active oxygen generating enzyme gene include a nitric oxide synthase gene and a xanthine oxidase gene. Nitric oxide synthase, which is a translation product of the nitric oxide synthase gene, and xanthine oxidase, which is a translation product of the xanthine oxidase gene, generate active oxygen. Active oxygen can be detected by an electron spin resonance apparatus (ESR apparatus) or the like. In the method using the ESR apparatus, the amount of active oxygen generated may be measured directly, or it may be measured using a specific spin trap agent. When a spin trap agent is used, first, the active oxygen generating enzyme is trapped by the spin trap agent, and then the amount of active oxygen generated from the trapped active oxygen generating enzyme is measured.

  A heavy metal binding protein gene may be used as a reporter gene. The heavy metal binding protein gene is a gene encoding a protein that specifically binds to a heavy metal. The amount of protein bound to the heavy metal may be determined by a magnetic resonance imaging apparatus, a nuclear medicine diagnostic apparatus, or an X-ray computed tomography apparatus. The amount of heavy metal binding protein generated is, for example, as follows. First, a measurable heavy metal is added in advance to a culture solution of a test cell. Next, after washing the test cells as necessary, a heavy metal binding protein is extracted from the test cells. Next, the extracted heavy metal binding protein is imaged with an image diagnostic apparatus corresponding to the heavy metal added to the culture solution, and the amount of heavy metal binding protein is measured. The heavy metal binding protein may be expressed inside the test cell or may be expressed on the outer surface of the test cell. An example of the heavy metal binding protein gene may be a base sequence encoding a metal compound binding peptide. For example, the metal compound binding protein may be a peptide, oligopeptide, polypeptide and / or protein that specifically binds to a specific metal compound. For example, the sequence encoding a peptide that binds to a metal compound may utilize the base sequence of an antibody gene or single chain antibody gene known to bind to the desired metal compound, and based on such base sequence Design. For such base sequences, for example, modifications and / or alterations such as substitution, deletion and addition of one or several bases within the range that maintains the binding with the metal compound, modifications according to the target to be applied, and You may design by modifying. A gene encoding a single-chain antibody peptide can be designed from the amino acid sequence of an antibody that binds a metal compound. For example, in MRI imaging, a gadolinium compound is preferably used because of its high contrast effect. The gadolinium compound may be a metal compound including, for example, gadolinium, gadolinium ions, gadolinium complexes, salts and derivatives thereof, derivatives containing any of these, or analogs of gadolinium compounds. It may also be a reporter gene that presents a metal compound binding ability outside the cell. Such a reporter gene may be a base sequence encoding a metal compound-binding peptide that is presented extracellularly. Such a base sequence is, for example, transcribed and translated in a cell to produce a metal compound-binding peptide, and the produced metal compound-binding peptide moves to the cell membrane and presents a metal compound-binding ability outside the cell. What is necessary is just to be comprised. Examples of such a reporter gene are disclosed in, for example, Japanese Patent Application Laid-Open No. 2012-200245.

  For example, a method for evaluating the metastatic property of a test cell using a self-replicating reporter vector includes preparing a reporter vector, introducing a reporter vector into the test cell, and expressing a replication initiator protein. Culturing the test cell under a certain condition and detecting the reporter protein expressed in the test cell may be included.

3-3. Kit for evaluating cell characteristics of test cells using reporter vector As a further embodiment, a kit for evaluating cell characteristics of test cells using a reporter vector may be provided. It may be a cell characterization kit for tumor cells. The cell property evaluation kit may include at least one reporter vector and a buffer among the reporter vectors described in 3-1. The cell property evaluation kit may further include a known carrier carrying a reporter vector and / or a container containing the reporter vector. Furthermore, the cell property evaluation kit may include any reagent necessary for performing a method for evaluating the cell property of a test cell, for example, a tumor cell. Such reagents may include, for example, reporter protein substrates produced from the reporter gene, excipients, stabilizers and / or other components having the desired effect. These cell property evaluation kits may be provided in a container.

4). Drug Screening Method A drug that suppresses abnormalities such as metastasis of a test cell may be screened using a method for evaluating cell characteristics of the test cell by detecting a NAMPT protein. Drugs that suppress tumor cell abnormalities, such as tumor cell metastasis, by detecting NAMPT protein may be screened. For example, if a drug-treated cell group treated with the drug to be screened and a pathological cell group not treated with the drug are prepared, cell characteristics are evaluated for each of these two cell groups, and the obtained results are compared. Good. The pathological cell group only needs to have an abnormality to be treated by the drug to be screened. Drug screening may be performed by using as an index whether such pathological cell characteristics have been treated by drug treatment. In addition to the drug-treated cell group and the pathological cell group, a normal cell group may be prepared and the cell characteristics of normal cells may be evaluated. The cellular properties of normal cells may be used as a control when performing drug screening. The cell characteristics of normal cells may be determined in parallel with drug screening, or may be determined in advance.

5. Cell characteristic evaluation apparatus for test cells The cell characteristic evaluation apparatus includes a gene introduction unit, a culture unit, an imaging unit, and a determination unit. The gene introduction unit is a unit that introduces a reporter vector containing a NAMPT gene promoter sequence into the nucleus of a test cell. The culture part is a unit that is processed by the gene introduction part, that is, a unit for culturing the test cell into which the gene has been introduced. The imaging unit captures a bright field image of the test cell cultured in the culture unit, and further captures a luminescence image composed of a luminescence signal generated for each cell derived from the expression of the reporter gene for the same field of view. Unit. The determination unit is a unit that determines cell characteristics of each cell of the test cell based on the bright field image and the light emission image.

  In a further example, the gene introduction part is a unit for introducing a reporter vector containing a NAMPT gene promoter sequence into the nucleus of a test cell. The culture part is a unit that is processed by the gene introduction part, that is, a unit for culturing the test cell into which the gene has been introduced. The imaging unit is a unit that captures a luminescence image composed of a luminescence signal generated for each cell derived from the expression of the reporter gene for the test cells cultured in the culture unit. A determination part is a unit which determines the cell characteristic for every cell of a test cell based on a luminescence picture.

  An example of an apparatus for determining cell characteristics of a test cell according to the embodiment will be described with reference to FIG. FIG. 3 is a block diagram showing an outline of the device 70. The apparatus 70 is an example of an apparatus that detects abnormal activity of a test cell and evaluates cell characteristics of the test cell based on the abnormal activity.

  The apparatus 70 includes a cell separation unit 71, a DNA introduction unit 72, a measurement unit 73, and a stage 74 arranged continuously below these. The cell separation unit 71, the DNA introduction unit 72, and the measurement unit 73 share one stage 74, and communicate with each other in this order by this stage 74. In other words, the cell separation unit 71, the DNA introduction unit 72, and the measurement unit 73 are arranged above the stage 74 in this order from upstream to downstream.

  The cell separation unit 71 includes a separator 75 for separating cells to be tested, that is, test cells, from a specimen obtained from a subject. The separator 75 separates a test cell from a receptor 76a that receives a sample obtained from a target, a processor 76b that digests the sample received by the receptor 76a, and a processed sample that has been processed by the processor 76b. And a separation unit 77. The receiving device 76a, the processing device 76b, and the separating portion 77 are formed in such a manner that three hollow cylindrical bodies having different inner diameters are stacked in this order from top to bottom, and are liquid-tightly integrated with each other. Each boundary portion between the receiving device 76a, the processing device 76b, and the separating portion 77 is liquid-tightly opened and closed by a valve. The receptacle 76a is a tapered cylindrical body having an open top and includes a lid that can be opened and closed. The valve is liquid tightly closed at the boundary between the receiver 76a and the processor 76b. When this valve is closed, it becomes the bottom of the receiver 76a. The processor 76b is a cylindrical body continuous with the lower end of the receiver 76a. When the valve at the boundary with the receiver 76a is liquid-tightly closed, the processor 76b is covered. When the valve at the boundary with the separation unit 77 is closed, the processor 76b is bottomed. The separation part 77 is a cylindrical body whose inner wall is liquid-tightly continuous from the inner wall of the processor 76b, and a valve is provided at the lower end thereof. The valve closes in a liquid-tight manner, and the separating portion 77 has a bottom. A separating material is fixed inside the separating portion 77. When the apparatus 70 is used, the container 78 is placed on the stage 74 below the bottom of the separation unit 77. The container 78 is a container 78 that receives and stores a sample including the separated test cell 79 from the separation unit 77. Here, the processor 76b may include a heating and / or thermostatic mechanism for performing a desired process such as digestion and biochemical reaction on the specimen contained therein. Further, the separation material may be a member that separates and collects a sample including the test cell from the sample that has fallen from the processing device 76b, or a contaminant from the sample that has fallen from the processing device 76b. It may be a member to be removed. The separating material may extend along a plane intersecting the central axis of the separating portion 77 and may be fixed over the circumference of the inner wall of the separating portion 77. The separating material may be, for example, a filter, a membrane, and / or a magnet.

  The sample including the test cell 79 separated by the separator 75 is accommodated in a container 78 placed on the stage 74. The container 78 that contains the test cells is sent to the DNA introduction unit 72.

  The DNA introduction unit 72 includes a control unit 80 and an introduction probe 81 for introducing a reporter vector into a test cell accommodated in a container 78. Introduction of a reporter vector into a test cell may be performed by a method known per se. The controller 80 and the introduction probe 81 may be any device that uses any introduction mechanism for achieving the selected introduction method. For example, the DNA introduction unit 72 may be a device that performs electroporation. In that case, the controller 80 is a DNA introducer, the introduction probe 81 is an electrode probe, and an electroporator may be constituted by these. Further, the DNA introduction unit 72 may include a nozzle for supplying a reagent containing a reporter vector to the container 78 (not shown). Further, the DNA introduction unit 72 may include a liquid feeding system for sending the reagent to the nozzle and a reagent storage container for supplying the reagent to the liquid feeding system (not shown).

  Further, the DNA introduction unit 72 may function as an incubator for culturing the test cell 79 in the container 78 under a predetermined culture condition for a predetermined time. The culture of the DNA introduction unit 72 may be controlled by the control unit 80.

  In the DNA introduction unit 72, the container 78 containing the test cells into which the reporter vector has been introduced is then sent to the measurement unit 73.

  The measurement unit 73 is installed in a dark box 82 that covers a part of the stage 74 from above and below, a light source 83 disposed in the dark box 82, a window that opens on a stage surface below the light source 83, and a window below the window. The imaging unit 88 to which the objective lens 87 is attached, a control unit 85 communicating with the light source 83 and the imaging unit 88, and a display unit 84 and a storage unit 86 communicating with the control unit 85, respectively. For example, the light source 83, the objective lens 87, and the imaging unit 88 of the measurement unit 73 may be included in a microscope equipped with a CCD camera or a CMOS camera. Moreover, the control part 85, the display part 84, and the memory | storage part 86 may be a computer provided with these.

  An apparatus for evaluating cell characteristics of test cells according to the embodiment will be described with reference to FIG. FIG. 4 is an example of an apparatus that differs from the apparatus 70 shown in FIG. 3 only in the configuration of the stage. The apparatus 70 includes a cell separation unit 71, a DNA introduction unit 72, a measurement unit 73, and stages 74a, 74b, and 74c disposed below the units. 3 is provided with a stage for each unit. Movement between the units, that is, between the stages may be performed manually, or each movement may be automatically performed by a robot arm or the like.

  One example of the evaluation of cell characteristics by the apparatus 70 shown in FIGS. 3 and 4 will be described with reference to FIG. First, the container 78 is placed on the stage 74 below the separation unit 77 of the cell separation unit 71. Next, a sample that has been pretreated with mince, homogenization, or the like as needed is brought into the receiver 76a from the upper opening. Along with this, reagents necessary for processing in the processor 76b are added. The addition of the reagent may be performed from a reagent addition port provided in the processor 76b so as to be opened and closed (S20). The lid of the receiver 76a is closed and the valve at the boundary with the processor 76b is opened. Digestion processing is performed in the processor 76b in accordance with preset conditions. The processor 76b may include a temperature controller for creating an environment that satisfies a preset condition (S21). Next, the valve at the boundary between the processor 76 b and the separation unit 77 is released, and the specimen processed in the processor 76 b is sent into the separation unit 77. By passing through the separation material of the separation unit 77, impurities contained in the specimen are removed (S22). The sample containing the separated test cells is sent to the container 78 and stored therein. Here, prior to passing through the separation material, a reaction stopping reagent for stopping the reaction advanced in the processor 76b may be added to the specimen. In this addition, a reaction stopping reagent may be stored in the separation unit 77 in advance, and an openable and closable reagent addition port for adding such a reagent is provided on the wall surface at any position of the separator 75. (S23).

  The container 78 is sent to the DNA introduction unit 72 while containing the sample including the test cell 79 (S24). A reagent containing a reporter vector is added to the container 78 from the nozzle of the DNA introduction unit 72. The introduction probe 81 is brought into contact with the sample in the container 78, and the reporter vector is introduced into the test cell 79 under the control of the control unit 80 (S25). Under the control of the control unit 80, the test cell 79 is cultured under a predetermined culture condition for a predetermined time (S26).

  Thereafter, the container 78 is sent to the measurement unit 73. In the measurement unit 73, the amount of reporter protein in the test cell 79 accommodated in the container 78 is measured. All procedures in the measurement unit 73 are controlled by the control unit 85 in accordance with a table preliminarily stored in the storage unit 86 and a table in which a plurality of pieces of information are collected. For example, the measurement unit 73 controls irradiation conditions such as light irradiation from the light source 83, irradiation time and irradiation interval, imaging and imaging condition control by the imaging unit 88, image processing and image processing for the captured image, and the like. Is done. The determination of cell characteristics is also performed by the control unit 85 with reference to a table including information in which images and cell characteristics are associated with each other in accordance with a program stored in the storage unit 86 in advance.

  After culturing the test cells in the DNA introduction unit 72, the container 78 is sent to the measurement unit 73. Thereafter, the light from the light source 83 is irradiated into the container 78. The light that has passed through the container 78 is collected by the objective lens 87, and the imaging unit 88 arbitrarily obtains a bright field image from the light. Thereafter, in the dark box 82, an optical signal (here, light emission as an example) from the reporter protein introduced into the test cell is imaged by the imaging unit 88 through the objective lens 87. Light emission from the light source 83 is stopped, light emission from the subject cell is collected by the objective lens 87, and the imaging unit 88 obtains a light emission image from the light (S27). Arbitrary bright field images and light emission images obtained by the imaging unit 88 are subjected to image processing by the control unit 85 and arbitrarily superimposed (merged) and displayed on the display unit 84. In the control unit 85, the cell characteristics of the test cell are determined based on the bright field image and the luminescent image and the table stored in the storage unit 86 at the same time as or before and after the display of the image on the display unit 84. judge. The result of this determination is displayed on the display unit 84 together with the merged image (S28).

  Here, an example in which light emission occurs as an optical signal from the reporter protein is shown, but for a detectable signal in which a reporter protein different from the light emission is generated, the detection device that constitutes the imaging unit 88 is replaced. It is possible to measure similarly.

  Here, although not shown in the figure, the measurement unit 73 may further include a processing unit that performs a computer process and an image process or an analysis unit that performs characteristic determination. These processing units and analysis units may perform the computer processing, image processing, and characteristic determination performed by the control unit 85 in the above description.

  In the above embodiment, the DNA introduction unit 72 performs the culture of the test cell. However, the test cell may be cultured in the measurement unit 73. In that case, the apparatus 70 may further include a member that enables cell culture. Alternatively, the test cells may be cultured in both the DNA introduction unit 72 and the measurement unit 73.

  In order to control the movement of each unit of the device 70, each unit may be provided with a control unit, and all the units included in the device 70 may be performed by one control unit, for example, the control unit 85. . Further, the movement between the units of the container 78 placed on the stage 74 may be performed by a container moving mechanism mounted on the stage 74. Such a mechanism may be performed by, for example, a robot arm that is provided in the device 70 and whose movement is controlled by a control unit provided in the device 70. Alternatively, a belt conveyor may be attached to the stage 74.

  The reporter vector may be added to the container 78 at any time before the container 78 is sent to a predetermined position of the DNA introduction unit 72. In that case, the apparatus 70 may further include a reporter vector addition mechanism at a desired position upstream from the DNA introduction unit 72.

  With such an apparatus, the cell characteristics of the test cells are evaluated. According to this apparatus, it is possible to evaluate the cell characteristics of the test cell with higher accuracy. In addition, since inspection can be performed with a throughput, it is possible to easily perform inspection, and it is possible to prevent sample mix-up and contamination.

  Further, morphological characteristics of the test cell may be obtained based on the bright field image, and the cell characteristics of the test cell may be evaluated together with information from the reporter vector. Morphological characteristics include, for example, the area of the test cell, the ratio of the area of the nucleus to the area of the test cell, the ratio of the short diameter to the long diameter of the test cell, the number of nuclei in the test cell or the side of the test cell It may be a feature of the edge, for example, a contrast ratio of the edge. These are obtained by non-invasive optical imaging methods. For example, these pieces of information may be obtained by hyperspectral imaging, phase contrast microscopy, differential interference, or the like.

  For example, when morphological observation is performed in a bright field, it is preferable to perform irradiation with light having a wavelength in the near infrared region. For example, the light having a wavelength in the near infrared region is preferably near infrared light having a wavelength of 700 nm to 750 nm or multispectral light of 350 nm to 1050 nm. For example, the test cell may be irradiated with light having a wavelength in the near-infrared region, and the reflection or absorption of the light, for example, the morphological information of the test cell may be obtained using the reflectance or absorption rate as an index .

  For example, when the test cell has a high metastatic property, the cell shape tends to be a cell shape having a high aspect ratio such as a spindle shape. For example, such morphological information of the test cell may be acquired in the measurement unit 73. Also in that case, the measurement unit 73 may be controlled by the control unit 85 according to a table in which a program stored in the storage unit 86 and a plurality of pieces of information are collected. The table contains information about the relationship between the detectable signal from the reporter vector and the cell characteristics, as well as the relationship between the morphological characteristics and cell characteristics of the test cell, the detectable signal and the cell characteristics. It is only necessary to include information indicating the relationship between and the morphological features. In addition, for example, the measurement unit 73 controls irradiation conditions such as light irradiation from the light source 83, irradiation time and irradiation interval, imaging and imaging condition control by the imaging unit 88, image processing and image processing on the captured image, and the like. Controlled by In addition, the evaluation of the cell characteristics is performed by referring to a table including information in which morphological characteristics, information from the reporter vector, and cell characteristics are associated with each other in accordance with a program stored in the storage unit 86 in advance. 85 may be performed. Thus, it is possible to obtain a more accurate result by evaluating the cell characteristics of the test cell from a plurality of aspects of promoter activity and morphological characteristics.

[Example]
As one example of a method for evaluating the cell characteristics of the test cells, the metastasis of tumor cell lines was evaluated. That is, a tumor cell line was used as an example of the test cell. Metastatic property was observed as an example of cell characteristics.

Example 1. Evaluation of metastasis of tumor cell lines by immunostaining (detection of NAMPT protein) For evaluation of metastasis, cell lines derived from human mammary tumors, BT-549 and MDA-MB-231, which are highly metastatic cell lines, and Low metastatic cell lines MCF-7 and T47-D were used. These cell lines were seeded on 8-well chamber slides (Thermo Scientific) and cultured at 37 ° C. in a 5% CO ₂ atmosphere. The medium was removed from the 8-well chamber slide, and the cells were washed with phosphate physiological buffer (PBS). Thereafter, 4% paraformaldehyde solution was added and incubated at room temperature for 20 minutes to immobilize the cells. After removing the paraformaldehyde solution and washing with PBS, 0.2% Triton X-100-containing PBS was added and incubated at room temperature for 10 minutes for membrane permeabilization. TritonX-100-containing PBS was removed, and after washing with PBS, Image-IT FX signal enhancer (Life Technologies) was added and incubated at room temperature for 30 minutes for blocking treatment. The Image-IT FX signal enhancer was removed and washed with PBS. Thereafter, Can Get Signal (TOYOBO) containing a rabbit anti-NAMPT polyclonal antibody (ab45890, Abcam) diluted 500 times was added and incubated overnight at 4 ° C. The antibody solution was removed, washed with PBS, Alexa546-labeled goat anti-rabbit antibody diluted 2000 times was added, and incubated at room temperature for 30 minutes. After removing the antibody solution and washing with PBS, the DAPI solution was added and incubated at room temperature for 1 minute to stain cell nuclei. After washing with PBS, the stained cells were encapsulated in a slide glass with Prolonged Gold Anti-Reagent (Life Technologies). The fluorescent image of the stained cells was observed with a microscope Axio Imager 2 (Carl Zeiss) and photographed. The fluorescence of Alexa 546 was observed with a filter set having an excitation wavelength of 550 nm and a fluorescence wavelength of 605 nm. The fluorescence of DAPI was observed with a filter set having an excitation wavelength of 320 nm and a fluorescence wavelength of 490 nm. The fluorescence image of the stained cells is shown in FIG.

  All breast tumor-derived cell lines subjected to the test were fluorescently stained with anti-Nampt protein. In the highly metastatic cell lines: BT-549 and MDA-MB-231, a stronger staining signal was detected than in the low metastatic cell lines: MCF-7 and T47-D. That is, the highly metastatic cell line highly expressed Nampt protein. From this result, it was shown that the metastatic property as an example of the cell characteristics of tumor cells can be evaluated using the same protein amount as an index.

Example 2. Evaluation of Metastasis Characteristics of Tumor Cell Lines by Reverse Transcription PCR (Detection of NAMPT mRNA) (1) Breast Tumor Cell Lines For cell lines derived from human mammary tumors, MDA-MB-231 and BT, which are highly metastatic cell lines, were used. -549, low metastatic cell lines MCF-7 and T47-D, and benign tumor cell line MCF-10A. These cell lines were seeded in 24-well plates and cultured at 37 ° C. in a 5% CO ₂ atmosphere. Total RNA was extracted from the cell line by RNeasy Mini Kit (Qiagen), and Nampt mRNA (mRNA transcribed from Nampt gene) was detected by RT-PCR (reverse transcription PCR).

  From the above-mentioned total RNA, cDNA was synthesized by a reverse transcription reaction using SuperScript VILO cDNA Synthesis Kit (Life Technologies). Thereafter, Nampt mRNA was amplified by PCR using the following primer set.

Forward primer; 5′-ACCCTTACAAGAGATTGTAG-3 ′ (SEQ ID NO: 2)
Reverse primer; 5′-TATGGAGAAGATCCTGACC-3 ′ (SEQ ID NO: 3)
After completion of PCR, the amplified product was electrophoresed on a 0.8% agarose gel, and the gel was stained with ethidium bromide. A photo was taken by visualizing the PCR amplification product in the gel under UV irradiation (FIG. 7). Here, all the photographs obtained by the above photographing have black backgrounds and white bands. If the image is shown in the drawing as it is, the band is difficult to see. In order to make the band easier to see, the image obtained by the above shooting is taken into a computer, and the image processing software is used to show an image with black and white colors reversed so as not to impair the band intensity and relativeness with other data. It was. NAMPT mRNA was detected in all breast tumor-derived cell lines subjected to the test. The highly metastatic cell lines MDA-MB-231 and BT-549 have a stronger amplification signal of NAMPT mRNA than the low metastatic cell lines MCF-7 and T47-D and the benign tumor cell line MCF-10A. was detected. That is, the highly metastatic cell line highly expressed NAMPT mRNA. From this result, it was shown that metastasis, which is an example of the cell characteristics, of a tumor cell, which is an example of a test cell, can be evaluated using the amount of NAMPT mRNA as an index.

(2) Lung tumor cell line In the experiment, A-549, which is a basal epithelial lung cancer cell line, and LUDLU-1, which is a squamous lung cancer cell line, were used as a lung tumor-derived cell line. Total RNA was extracted from these cell lines in the same manner as in Example (1) above, and NAMPT mRNA was detected by RT-PCR. The primer sets described in SEQ ID NOs: 2 and 3 were used as PCR primers.

  After completion of PCR, the amplified product was electrophoresed on a 0.8% agarose gel, and the gel was stained with ethidium bromide. A photo was taken by visualizing the PCR amplification product in the gel under UV irradiation (FIG. 8). Here, all the photographs obtained by the above photographing have black backgrounds and white bands. If the image is shown in the drawing as it is, the band is difficult to see. In order to make the band easier to see, the image obtained by the above shooting is taken into a computer, and the image processing software is used to show an image with black and white colors reversed so as not to impair the band intensity and relativeness with other data. It was. NAMPT mRNA was detected in any cell line of A-549, a basal epithelial lung cancer cell line, and LUCLU-1, a squamous lung cancer cell line. Further, it was revealed that A-549, which is a low metastatic cell line, has a small amount of NAMPT mRNA.

Example 3 Evaluation of tumor cell metastasis by reporter gene assay (detection of NAMPT promoter activity) (1) Breast cancer cell line (a) Construction of reporter vector Promoter region of nicotinamide phosphoribosyltransferase (Nampt) gene (−2426 to +276 bp, SEQ ID NO: A reporter vector incorporating 1) was prepared. The promoter region of the Nampt gene was amplified by PCR using a human genome as a template. PCR was performed using PrimeStar GLX DNA polymerase (TaKaRa BIO). The primer sequences used for PCR are shown below.

Forward primer; 5′-GGGACGCGTCCATGTTGCCCAGCTGGTCTCA-3 ′ (SEQ ID NO: 4)
Reverse primer; 5′-CGGCTCGAGAGCTCCCTGGCGCGCGCTGCAGGAA-3 ′ (SEQ ID NO: 5)
The promoter sequence obtained by PCR was incorporated into PGV-B2 (TOYO B-Net) to prepare a reporter vector: pNampt-L. PGV-B2 is a vector comprising a luciferase gene (reporter gene) and a transcription termination signal sequence in this order from upstream to downstream. Specifically, a CK19 promoter region, which is a promoter sequence amplified by PCR, was incorporated upstream of the PGV-B2 luciferase gene to prepare a reporter vector: pNampt-L (FIG. 9).

  PNLuc-tk was used as a vector for correcting the introduction efficiency of the reporter vector for each cell line. pNLuc-tk was prepared by incorporating the human herpesvirus thymidine kinase (tk) gene promoter of pRL-tk (Promega) upstream of the NanoLuc gene of pNL1.1 (Promega).

(B) Lipofection (introduction of reporter DNA into cells)
To 40 μl of Opti-MEM (Life Technologies), 0.15 μg of a reporter vector: pNampt-L and 0.05 μg of a vector for correcting introduction efficiency: pNLuc-tk were added. Thereafter, 0.2 μg of enhancer reagent (Plus reagent, Life Technologies) was added and incubated at room temperature for 5 minutes. To this solution, 0.7 μl of Lipofectamine LTX (Life Technologies) was added and well suspended, and incubated at room temperature for 25 minutes. Transfer the solution to a 48-well plate and add 2.0 ml of 5 cell suspensions derived from human mammary tumors (MDA-MB-231, BT-549, MCF-7, T-47D and MCF-10A) And gently mixed. Thereafter, the cells were cultured in an incubator at 37 ° C. and 5% CO ₂ atmosphere.

(C) Reporter gene assay 72 hours after the lipofection in Example 3 (b) above, the culture solution was removed from the 48-well plate and washed with PBS. Thereafter, 150 μl of cell lysate (Glo-Lysis Buffer) (Promega) was added, and the mixture was allowed to stand at −80 ° C. for 30 minutes or more and frozen. At the time of use, after thawing the cell lysate at room temperature, the lysate was collected in a centrifuge tube, and cell debris was precipitated by centrifugation. The supernatant was collected in a new sample tube, and 25 μl of the supernatant was dispensed into a 96-well plate (Nunc). After adding an equal amount of Luciferase substrate solution (One-Glo Luciferase Assay System, Promega) or NanoLuc substrate solution (Nano-Glo Luciferase Assay System) (Promega) to L, 40 μm, L Was measured. Thereby, the activity of pNampt-L and the activity of reporter DNA for correcting the introduction efficiency: pNLuc-tk were measured. The relative luminescence intensity of pNampt-L was a value obtained by dividing the activity value (luminescence intensity: RLU) of the reporter DNA by the activity value (luminescence intensity) of pNLuc-tk. The results are shown in FIG. BT-549, a highly metastatic cell line, showed stronger luminescence intensity (RLU) than MCF-7 and T47-D, which are low metastatic cell lines, and MCF-10A, which is a benign tumor cell line. That is, it was revealed that the highly metastatic cell line has high NAMPT promoter activity. Thus, it was shown that metastasis, which is an example of cell characteristics, can be evaluated for tumor cells, which are examples of test cells, using NAMPT promoter activity as an index.

(2) Lung cancer cell line (a) Reporter vector The following experiment was conducted using pNampt-L described in Example 3 (1) (a) above.

(B) Lipofection (introduction of reporter DNA into cells)
Into two types of cell lines derived from human lung cancer, A-549 and LUDLU-1, pNampt-L was introduced into the cells by the same lipofection method as in Example 3 (1) (b) above.

(C) Reporter gene assay A cell lysate was prepared in the same manner as in Example 3 (1) (c) above. Thereafter, cell debris is precipitated by centrifugation, and 25 μl of the supernatant is dispensed into a 96-well plate (Nunc), and an equal amount of Luciferase substrate solution (One-Glo Luciferase Assay System, Promega) or NanoLuc substrate solution (Nano- Glo Luciferase Assay System (Promega) was added, and the luminescence intensity was measured with a luminometer (Mithras LB940, Berthold). The relative luminescence intensity of pNampt-L was obtained by dividing the activity value (luminescence intensity) of the reporter DNA by the activity value (luminescence intensity) of pNLuc-tk. The results are shown in FIG. Both A-549, a basal epithelial lung cancer cell line, and LUCLU-1, a squamous lung cancer cell line, showed luminescence. That is, NAMPT promoter activity was detected from both cell lines. Further, it was revealed that A-549, which is a low metastatic cell line, has low NAMPT promoter activity.

Example 4 Detection of metastatic breast cancer cell line by cell luminescence imaging (1) Reporter vector The reporter vector prepared in Example 3 (1): pNampt-L was used in the experiment.

(2) Lipofection (introduction of reporter DNA into cells)
To 500 μl of Opti-MEM (Life Technologies), 2.5 μg of reporter vector: pNampt-L and 2.5 μg of enhancer reagent (Plus reagent, Life Technologies) were added. This was incubated at room temperature for 5 minutes. To this solution, 6.25 μl of Lipofectamine LTX (Life Technologies) was added and well suspended. Thereafter, it was incubated at room temperature for 25 minutes. The solution was transferred to a 35 mm culture dish, and BT-549, a high metastatic malignant tumor cell line derived from human breast tumor, MCF-7, a low metastatic malignant tumor cell line, and a benign tumor cell line derived from human mammary tumor Each 2.0 ml of MCF-10A suspension was added and gently mixed. Thereafter, the cells were cultured in an incubator at 37 ° C. and 5% CO ₂ atmosphere.

(3) Luminescence Imaging Luminescence imaging of a reporter vector-introduced cell line was performed with a LUMINOWEW luminescence imaging system (LV200, Olympus). 24 hours after lipofection, the luminescent substrate VivoGloluciferin (Promega) was added to the medium of BT-549, MCF-7, and MCF-10A into which pNampt-L was introduced (final concentration 200 μM). The culture dish was set at a predetermined position of LUMINOWEW, and microscopic images (× 20 times) of the cells were taken in bright field and dark field (luminescence). The shooting in the dark field was performed with an exposure time (light emission detection time) of 20 minutes. The results of luminescence imaging are shown in FIG. FIG. 12 shows a superimposed image of a bright field image and a dark field image (light emission image). Image superposition was performed with image processing software METAMORPH. Strongly luminescent cells were observed in the highly metastatic malignant tumor cell BT-549 (FIG. 12 (a), BT-549). Luminescent cells were observed in the low-metastatic tumor cell MCF-7 (FIG. 12 (b), MCF-7), but the luminescence was weak. In addition, no luminescent cells were observed in the benign tumor cell MCF-10A (FIG. 12 (c), MCF-10A).

  According to these embodiments or examples, a new technique for evaluating cell characteristics can be provided.

  Although several embodiments of the present invention have been described, these embodiments are presented by way of example and are not intended to limit the scope of the invention. These novel embodiments can be implemented in various other forms, and various omissions, replacements, and changes can be made without departing from the scope of the invention. These embodiments and modifications thereof are included in the scope and gist of the invention, and are included in the invention described in the claims and the equivalents thereof.

Claims (27)

  A gene marker for evaluating cell characteristics of a test cell, wherein the gene marker is a mRNA containing a NAMPT gene mRNA, peptide or protein, or a NAMPT gene promoter sequence.   The genetic marker according to claim 1, wherein the test cell is an epithelial tumor.   The genetic marker according to claim 1, wherein the test cell is a tumor cell derived from a mammary gland tissue or a lung tissue.   A method for evaluating cell characteristics of a test cell, characterized by using the expression level of the NAMPT gene in the test cell as an index.   The method according to claim 4, wherein the test cell is an epithelial tumor.   The method according to claim 4, wherein the test cells are tumor cells derived from mammary gland tissue or lung tissue. Using the expression level of the NAMPT gene as an index,
Measuring the expression level of the NAMPT gene in the test cell;
Comparing the measured expression level of the test cells with a threshold value;
The method includes: determining a degree of expression of a NAMPT gene in the test cell based on the comparison result; and evaluating cell characteristics of the test cell based on the determination. The method according to any one of 4 to 6.   The method according to claim 7, wherein the threshold is determined based on an expression level of a NAMPT gene from healthy cells. The expression level of the NAMPT gene from the healthy cells is
Obtained by measuring the expression level of the NAMPT gene in the healthy cells prior to the measurement of the expression level of the test cells,
Obtained by measuring the expression level of the NAMPT gene in the healthy cells after the measurement of the expression level of the test cells, or in parallel with the measurement of the expression level of the test cells, The method according to claim 8, wherein the method is obtained by measuring the expression level of.   The method according to any one of claims 7 to 9, wherein the expression level of the NAMPT gene is measured by measuring a NAMPT protein mass, a NAMPT mRNA level, or a NAMPT gene promoter activity level.   The method according to claim 10, wherein the measurement of the amount of NAMPT protein is performed by an immunological assay using an anti-NAMPT antibody.   The immunological measurement method is enzyme immunization (EIA), fluorescence immunization (FIA), automatic cell analysis / separation device (FACS), immune cell staining (ICC) or immunohistochemical staining (IHC) The method of claim 11, wherein the method is characterized in that:   The method according to claim 10, wherein the measurement of the amount of NAMPT mRNA is performed by a reverse transcription PCR method.   In the reverse transcription PCR method, an amplification reaction is carried out using a primer set comprising a first primer represented by SEQ ID NO: 2 or a complementary sequence thereof and a second primer represented by SEQ ID NO: 3 or a complementary sequence thereof The method according to claim 13.   The method according to claim 10, wherein the measurement of the NAMPT gene promoter activity is performed using a reporter vector containing a NAMPT gene promoter sequence. The reporter vector comprises a reporter gene expression unit;
The reporter gene expression unit is
(Aa) the 5 ′ upstream promoter sequence of the NAMPT gene;
(Ab) a reporter gene encoding a detectable reporter protein operably linked downstream of the promoter sequence; and (Ac) a transcription termination signal sequence operably linked downstream of the reporter gene. ;
The method according to claim 15, comprising: The reporter vector further comprises a replication initiation unit linked on the same nucleic acid as the reporter gene expression unit, the replication initiation unit comprising:
(B) a replication initiation sequence that initiates self-replication of the reporter vector upon binding of a replication initiator protein;
Measuring the expression level of the NAMPT gene,
(1) preparing the reporter vector;
(2) introducing the reporter vector into the test cell;
(3) culturing the test cell under conditions where the replication initiation protein is expressed:
(4) detecting the reporter protein expressed in the test cell;
The method of claim 16 comprising: A reporter vector for evaluating cell characteristics of a test cell, wherein the reporter vector includes a reporter gene expression unit, and the reporter gene expression unit comprises:
(Aa) the 5 ′ upstream promoter sequence of the NAMPT gene;
(Ab) a reporter gene encoding a detectable reporter protein operably linked downstream of the promoter sequence; and (Ac) a transcription termination signal sequence operably linked downstream of the reporter gene. ;
A reporter vector comprising: The reporter vector further comprises a replication initiation unit and a replication initiation protein unit linked on the same nucleic acid as the reporter gene expression unit, and the replication initiation unit comprises:
(B) a replication initiation sequence that initiates self-replication of the reporter vector upon binding of a replication initiator protein;
The replication initiator protein unit comprises:
(Ca) an IRES sequence operably linked downstream of the reporter gene; and (Cb) a replication initiator protein gene encoding a replication initiator protein operably linked downstream of the IRES sequence;
Including
19. The reporter vector according to claim 18, wherein the transcription termination signal sequence (Ac) is operably linked downstream of the replication initiation protein gene (CB).   The reporter vector according to claim 18, wherein the IRES sequence is an encephalomyocarditis virus IRES sequence. The reporter vector further comprises a replication initiation unit and a replication initiation protein unit linked on the same nucleic acid as the reporter gene expression unit, and the replication initiation unit comprises:
(B) a replication initiation sequence that initiates self-replication of the reporter vector upon binding of a replication initiator protein;
The replication initiator protein unit comprises:
(Da) a constitutive promoter that is constitutively expressed;
(Db) a replication initiator protein gene encoding the replication initiator protein operably linked downstream of the constitutive promoter; and (Dc) operably linked downstream of the replication initiator protein gene. A further transcription termination signal sequence;
The reporter vector according to claim 18, comprising:   The replication origin protein gene is a large T antigen gene of simian virus 40, and the replication initiation sequence is a replication initiation sequence of simian virus 40. Reporter vector.   The reporter vector according to any one of claims 18 to 22, wherein the 5 'upstream promoter sequence of the NAMPT gene comprises a sequence represented by SEQ ID NO: 1.   The reporter vector according to any one of claims 18 to 23, wherein the reporter gene is a luminescent gene, a fluorescent gene, a chromogenic gene, a heavy metal binding gene, or a nitric oxide synthesis gene.   A primer set for evaluating the cell characteristics of a test cell, comprising a first primer represented by SEQ ID NO: 2 or a complementary sequence thereof, and a second primer represented by SEQ ID NO: 3 or a complementary sequence thereof Primer set. An assay kit for evaluating cell characteristics of a test cell,
(1) The reporter vector and buffer according to any one of claims 18 to 24; or (2) the primer set and buffer according to claim 25;
An assay kit comprising: (1) a gene introduction part for introducing a reporter vector containing a NAMPT gene promoter sequence into the nucleus of a test cell;
(2) a culture unit for culturing the test cells treated in the gene introduction unit;
(3) Taking a bright field image of the test cell cultured in the culture section, and further taking a luminescence image consisting of a luminescence signal generated for each cell from the expression of the reporter gene in the same field An imaging unit to
(4) A cell characteristic evaluation apparatus comprising: a determination unit that determines cell characteristics of each cell of the subject cell based on the bright field image and the light emission image.

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