JP2004512014A - Testing method for ischemic condition - Google Patents

Abstract

Examination of ischemic condition by measuring the expression level of a specific gene in a test sample or determining the expression profile of a group of genes containing any of the specific gene Method.

Description

【００７２】
【表２】

【００７３】
【表３】

【００７４】
本明細書中引用した文献、特許及び特許出願は全て参照によりその全体を本明細書に組み入れるものとする。
【００７５】
産業上の利用可能性
被検試料中の遺伝子発現を解析することにより、虚血状態が検査される。このような解析は、虚血の予防及び治療への応用が期待される。また、虚血状態に特異的に発現される遺伝子の発現レベルを指標とする、新規な虚血予防薬及び治療薬のスクリーニング方法が提供される。
【００７６】
配列表フリーテキスト
配列番号１０６７：合成ＤＮＡ
配列番号１０６８：合成ＤＮＡ

【図面の簡単な説明】
【図１】
図１は、ＤＮＡチップを用いて、虚血状態において発現レベルの変動する遺伝子を同定する手順を示した図である。
【図２】
図２は、本発明の虚血状態検査用ＤＮＡチップの一態様及び当該ＤＮＡチップを用いて被検患者由来の試料をハイブリズさせたときに予想される結果を示した図である。
【図３】
図３は、合成型ＤＮＡチップの一態様及び当該ＤＮＡチップを用いて被検試料中の遺伝子を検出する手順を示した図である。
【図４】
図４は、ＧｅｎｅＣｈｉｐ^ＴＭを用いて遺伝子発現レベルを測定する場合の手順を示した図である。
【図５】
図５は、虚血状態同定システムのブロック図である。
【図６】
図６は、虚血状態同定プログラムによる処理例を示すフローチャートの図である。
【符号の説明】
５０１：ＣＰＵ、 ５０２：ＲＯＭ、　５０３：ＲＡＭ、　５０４：入力部、　５０５：送信／受信部、　５０６：出力部、　５０７：ＨＤＤ、　５０８：ＣＤ−ＲＯＭドライブ、　５０９：ＣＤ−ＲＯＭ、　５１０：データベース　６０１：クラスター分析装置、　６０２：外部データベース検索入力手段、　６０３：サンプルデータ記憶手段、　６０４：データ最適化手段、　 ６０５：変量一覧出力手段、　６０６：変量選択手段、　６０７：評価用サンプルデータファイル生成手段、　６０８：評価手段、　６０９：クラスター分類手段、　６１０：樹形図生成手段、　６１１：樹形図編集手段、　６１２：評価条件設定手段[0001]
Technical field
The present invention provides an ischemic condition by measuring the expression level of a specific gene in a test sample or determining the expression profile of a group of genes including a plurality of genes selected from the specific gene in the sample. The present invention relates to a method for examining an ischemic state, characterized by examining.
[0002]
Background art
Cancer, stroke and heart disease are called the three major adult diseases, and these three accounts for about 60% of Japanese deaths. Of these, stroke and heart disease are often caused by ischemia. Therefore, the disease can be prevented beforehand by detecting the ischemic state early. Ischemia is a localized anemia. Due to its mechanism, intravascular or vascular itself such as compressive ischemia or thrombosis / arteriosclerosis caused by stenosis or occlusion of the arterial wall due to external compression such as a tumor. Cerebral anemia and convulsive ischemia due to vasospasm, such as cerebral anemia and angina. Ischemia in the brain triggers ischemic stroke such as cerebral infarction, and ischemia in the heart triggers ischemic heart disease such as myocardial infarction. Therefore, for the prevention of the above-mentioned diseases, first and foremost, it is important to detect the ischemic state as soon as possible and to take appropriate measures.
[0003]
Conventionally, as a method for examining the ischemic state, a method using cardiac wall motion abnormality as an index (for example, quantitative analysis of ventricular morphology / ultrasound image, detection of decrease in tissue contraction rate, etc.), and an abnormal hemodynamic index as an index Methods (eg, analysis of left ventricular inflow rate patterns, nuclear medicine tests, etc.) are known. Among them, the nuclear medicine test method is a method capable of precisely examining the ischemic state. However, there were many disadvantages for the subject such as radiation exposure, long examination time and high cost. Therefore, there has been a demand for a simple ischemic test method which can be performed on a daily basis as a part of a health examination with a small burden on the subject.
[0004]
Disclosure of the invention
The present invention is characterized by easily examining the ischemic state by measuring the expression level of a specific gene in a test sample or the expression profile of a group of genes including a plurality of genes selected from the specific gene. It is an object of the present invention to provide a test method for an ischemic condition.
[0005]
The present inventors have conducted intensive studies to solve the above-mentioned problems, and as a result, have successfully identified a gene expressed in an ischemic state, clarified the gene expression profile, and completed the present invention. Was.
[0006]
That is, the present invention provides an ischemic condition by measuring the expression level of a specific gene in a test sample or determining the expression profile of a group of genes including a plurality of genes selected from the specific gene in the sample. Is an ischemic condition inspection method. Here, as the specific gene, (a) a gene having any of the nucleotide sequences shown in SEQ ID NOS: 1 to 1066 or a gene encoding any of the amino acid sequences shown in SEQ ID NOs: 1 to 1066 Or (b) a gene functionally equivalent to a gene having any of the above nucleotide sequences, or a gene functionally equivalent to a gene encoding any of the above amino acid sequences. The measurement of the expression level or the determination of the expression profile can be performed on a DNA chip (for example, a synthetic DNA chip). The ischemia includes compressional ischemia, obstructive ischemia, spastic ischemia and the like.
[0007]
Furthermore, the present invention provides (a) a gene having any one of the nucleotide sequences shown in SEQ ID NOs: 1 to 1066, or a gene encoding any of the amino acid sequences shown in SEQ ID NOs: 1 to 1066, or (b) 2.) Some or all of a gene functionally equivalent to a gene having any of the above nucleotide sequences or a gene functionally equivalent to a gene encoding any of the above amino acid sequences was immobilized on its surface. It is a DNA chip for ischemic condition inspection. The ischemic condition includes compressional ischemia, obstructive ischemia, spastic ischemia and the like.
[0008]
Furthermore, the present invention is a method for screening an ischemic condition ameliorating agent or a therapeutic agent for ischemic disease. The method comprises the steps of: (a) administering an agent to a test animal or a test cell; Whether or not the expression level of the fluctuating specific gene returns to a normal expression level, or (b) whether or not the expression profile of a group of genes including a plurality of the specific gene returns to a normal expression profile. The method is characterized in that a candidate drug is selected as an index, and a return to a normal expression level or a normal expression profile indicates that the drug is a candidate drug. Here, as the specific gene, (a) a gene having any of the nucleotide sequences shown in SEQ ID NOS: 1 to 1066 or a gene encoding any of the amino acid sequences shown in SEQ ID NOs: 1 to 1066 Or (b) a gene functionally equivalent to a gene having any of the above nucleotide sequences, or a gene functionally equivalent to a gene encoding any of the above amino acid sequences. The ischemic condition includes compressional ischemia, obstructive ischemia, spastic ischemia and the like.
[0009]
Further, the present invention provides (i) expression level data of a specific gene whose expression level fluctuates in an ischemic state, or (ii) expression profile data of a gene group including a plurality of genes selected from the specific gene, Is a computer-readable recording medium on which is recorded. Here, as the specific gene, (a) a gene having any of the nucleotide sequences shown in SEQ ID NOS: 1 to 1066 or a gene encoding any of the amino acid sequences shown in SEQ ID NOs: 1 to 1066 Or (b) a gene functionally equivalent to a gene having any of the above nucleotide sequences, or a gene functionally equivalent to a gene encoding any of the above amino acid sequences. The ischemic condition includes compressional ischemia, obstructive ischemia, spastic ischemia and the like.
[0010]
Furthermore, the present invention provides (a) a procedure for inputting expression level data or expression profile data of a specific gene in a test sample, (b) a procedure for recording the input data, and (c) a procedure for recording the already recorded data. A procedure for collating the recorded expression level data or expression profile data of the gene in the ischemic state, and determining whether the test sample is in an ischemic state based on the collation results in (d) and (c). And (e) when the test sample is in an ischemic state, the procedure for causing the computer to identify the ischemic clinical stage of the test sample based on the collation result in (c). It is a computer-readable recording medium that has been recorded. Here, as the specific gene, (a) a gene having any of the nucleotide sequences shown in SEQ ID NOS: 1 to 1066 or a gene encoding any of the amino acid sequences shown in SEQ ID NOs: 1 to 1066 Or (b) a gene functionally equivalent to a gene having any of the above nucleotide sequences, or a gene functionally equivalent to a gene encoding any of the above amino acid sequences. The ischemia includes compressional ischemia, obstructive ischemia, spastic ischemia and the like.
[0011]
Hereinafter, the present invention will be described in detail.
[0012]
This application claims the priority of Japanese Patent Application No. 2000-145977, and the contents of the specification and drawings of the application are included in the present specification.
[0013]
The present invention relates to an ischemic condition test method characterized by examining an ischemic condition using an expression level of a specific gene or an expression profile of a specific gene group as an index, which is different from a conventional ischemic test method. Here, the “expression level” refers to the absolute amount or relative amount of a transcript (mRNA) of a specific gene or the absolute amount or relative amount of a translation product (protein) of a specific gene. The “expression profile” refers to a table or graph that summarizes the expression levels of a plurality of genes.
[0014]
1. Identification of genes whose expression levels fluctuate in ischemic conditions
Genes whose expression levels fluctuate in the ischemic state can be determined, for example, by a differential RNA display method [Liang, Pet. : Science, 257: 967-971 (1992)], a hybrid subtraction method, a method using a DNA chip, and the like. For example, a method for identifying the gene using a DNA chip can be performed as shown in FIG. That is, a plurality of pieces of DNA information (cDNA / EST / oligo DNA collection) are obtained from a database in which a genomic sequence, a cDNA sequence, an EST sequence, or the like is recorded, and various genes whose base sequences are apparent are fixed on a DNA chip. . Thereafter, a labeled DNA or RNA prepared based on mRNA derived from an ischemic biological sample is hybridized to the DNA chip. Thereafter, the expression level of each gene is measured by measuring the hybridization intensity at each spot of the obtained hybridization pattern to obtain an expression profile. Hereinafter, a method using a DNA chip will be described in more detail.
[0015]
(1) Preparation of poly (A) + mRNA from test sample
In order to examine the expression level of a certain gene in a biological sample using a DNA chip, first, it is necessary to prepare poly (A) + mRNA from a test sample such as a tissue or a cell. As a specific example of a test sample for preparing poly (A) + mRNA for identifying a gene whose expression level fluctuates in an ischemic state, an experimental animal (eg, mouse , Rats, guinea pigs, rabbits, dogs, cats, pigs, cows) or ischemic human-derived biological tissues (eg, blood tissues, brain tissues, heart tissues, kidney tissues). In the brain, it is said that about 80% of genes that can be expressed in vivo are expressed. Therefore, by examining genes whose expression levels fluctuate in the brain in an ischemic state, it is possible to comprehensively identify genes whose expression levels fluctuate in an ischemic state in tissues other than the brain. The hippocampus derived from the mouse can be used as a sample for preparing poly (A) + mRNA. Blood cells such as red blood cells, white blood cells, and platelets are mixed in the hippocampus due to the presence of brain capillaries. Therefore, poly (A) + mRNA extracted from hippocampal tissue may contain mRNAs derived from various blood cells.
[0016]
The preparation of poly (A) + mRNA was performed by the guanidine thiocyanate-cesium chloride method [J. See Sambrook, et al. , Molecular Cloning, 2^nd Ed. , Cold Spring Harbor Laboratory Press, New York (1989)], the guanidine thiocyanate-cesium trifluoroacetate method [H. Okayama, et al. , Methods in Enzymology, 154: 3, Academic Press, New York (1987)], guanidine thiocyanate-phenol-chloroform method [P. See Chomczynski et al. , Anal. Biochem. 162: 156 (1987)], phenol-SDS method [R. D. Palmiter, Biochemistry, 13: 3606 (1974)] and the like, and the obtained total RNA is subjected to an oligo dT cellulose or poly-U Sepharose column, and after specifically adsorbing poly (A) + mRNA. , Poly (A) + mRNA from the column. In particular, when using a tissue as a test sample, the purification state of total RNA or poly (A) + mRNA greatly affects the yield of cDNA and the like, so it is important that the purification process be performed accurately.
[0017]
For example, when preparing poly (A) + mRNA by the guanidine thiocyanate-cesium chloride method, first, an appropriate amount (for example, 5 times) of a guanidine thiocyanate solution is added to a tissue sample. Next, the tissue sample is crushed by a homogenizer such as Polytron, and then N-lauroyl sarcosinate is added to a desired concentration (for example, 0.5%), followed by stirring. The obtained sample was centrifuged (for example, 5000 × g, 10 minutes), and the supernatant was layered on a centrifuge tube containing a cushion of cesium chloride-EDTA, followed by ultracentrifugation (for example, 100,000 × g, 12 g). Time). After rinsing the precipitate with 70% ethanol, the precipitate is dissolved in TE buffer to obtain total RNA. Poly (A) + mRNA can be obtained by subjecting the obtained total RNA to an oligo dT cellulose column.
[0018]
The preparation of poly (A) + mRNA can also be performed using a commercially available kit. Specific examples of the kit for preparing total RNA include RNeasy Total RNA Isolation kit (manufactured by QIAGEN) and TRIzol Reagent (manufactured by Gibco BRL Life Technologies). Specific examples of the kit for isolating poly (A) + mRNA from total RNA include Oligotex Direct mRNA Kit (QIAGEN), Oligotex mRNA Kit (QIAGEN) and the like.
[0019]
(2) cDNA synthesis by reverse transcriptase
Next, cDNA is synthesized using the poly (A) + mRNA obtained in the above (1) as a template. cDNA synthesis was performed according to the method of Gubler et al. [U. Gubler, et al. , Gene, 25: 263 (1987)]. That is, first, oligo (dT) is added to the poly (A) + mRNA solution._12-18, And then rapidly cooled after heating. Next, a buffer solution for single-stranded cDNA synthesis, a dNTP (mixture of dATP, dGTP, dCTP, and dTTP) solution, a ribonuclease inhibitor solution, a dithiothreitol solution, and the like are added to the solution, and the mixture is mixed. (For example, Superscript RT (manufactured by BRL)) is added, and the mixture is incubated for a certain period of time to obtain a single-stranded cDNA. Further, if necessary, a double-stranded cDNA can be synthesized using the obtained single-stranded cDNA as a template. That is, a buffer solution for cDNA synthesis, a dNTP (mixture of dATP, dGTP, dCTP, and dTTP) solution, a dithiothreitol solution, and the like are added to the single-stranded cDNA solution and mixed, and then a DNA polymerase (eg, T4 DNA polymerase) is added to the mixture. Then, double-stranded cDNA can be obtained by incubating for a certain period of time. In the synthesis of single-stranded or double-stranded cDNA, a labeled cDNA can be obtained by using a labeled dNTP (for example, biotin-labeled dNTP).
[0020]
(3) Preparation of labeled cRNA fragment
In the method of the present invention, when a DNA chip on which an oligonucleotide is immobilized is used as a DNA probe, if necessary, the cRNA obtained in the above (2) may be used as a template, and the labeled cRNA may be further subjected to in vitro transcription. Is prepared. Preparation of labeled cRNA by in vitro transcription is performed according to the method of Kreig et al. [Kreig, P .; A. , Et al. , Methods in Enzymology, 155: 397-415 (1987)]. Note that the obtained labeled cRNA molecule needs to be reduced (fragmented) before use. Low molecular weight is Mg²⁺Heating in the presence (for example, 94 ° C. for 3 minutes) and DNase treatment can be performed.
[0021]
The above-mentioned in vitro transcription can also be performed using a commercially available kit. MEGAscript is used as an in vitro transcription kit.^TM In Vitro Transcription Kits (manufactured by Ambion) and the like.
[0022]
(4) Hybridization on DNA chip
Next, a hybridization reaction is performed by injecting the labeled nucleotide sample obtained in the above (2) or (3) into a DNA chip. Specific examples of a DNA chip useful in the method of the present invention include an oligo DNA microarray (also referred to as a synthetic DNA chip) prepared by directly synthesizing an oligo DNA on a substrate, and immobilizing DNA prepared in advance on the substrate. DNA microarray (also referred to as an attached DNA chip). In the present invention, in order to identify a gene whose expression level fluctuates in an ischemic state, a synthetic DNA chip capable of obtaining high detection sensitivity, accuracy, and reproducibility, for example, an oligo DNA microarray (GeneChip, manufactured by Affymetrix, Inc.)^TM) Is preferably used.
[0023]
When examining gene expression, it is important to perform hybridization under conditions of high stringency in order to suppress non-specific binding. Here, “high stringency conditions” refers to conditions under which hybridization occurs only between nucleotide chains having 90% or more homology. The level of stringency can be adjusted by changing the salt concentration (eg, NaCl, trisodium citrate, etc.) and / or temperature; the lower the salt concentration and the higher the temperature, the higher the stringency. Become. Certain temperature and salt concentration conditions can act as either high stringency or low stringency conditions, depending on the type of DNA chip used and other factors. Therefore, what condition is a condition of high stringency and similarly what condition is a condition of low stringency should be determined for each chip to be used. GeneChip used in the present invention^TM In the case of Mu6500, high stringency conditions include a temperature of 43 to 65 ° C., preferably 45 ° C.⁺The concentration refers to a condition of 500 to 1000 mM, preferably 1000 mM.
[0024]
(5) Detection and data analysis
As a result of the hybridization, the double strands formed on the microarray are analyzed with a fluorescence image scanner or the like. The fluorescence intensity on the microarray can be automatically measured by a system in which a fluorescence laser microscope, a CCD camera, and a computer are connected. The scanner is preferably one capable of quantitatively identifying spots having a size of several tens of μm and a distance between the spots of about 10 μm. Further, it is preferable that the scanner be capable of coping with a plurality of types of markers, capable of scanning a wide range at high speed, and having an autofocus function capable of coping with microscopic distortion of a substrate. Examples of the scanner having such a function include a GMS 418 Array Reader (manufactured by Genetic Micro Systems). Further, the software used for data analysis is preferably one that can cope with complicated analysis including a large number of oligonucleotides having partially overlapping sequences, such as mutation or polymorphism analysis.
[0025]
In the present invention, it is possible to use a commercially available system in which a set of components necessary for gene analysis using a DNA chip is incorporated. Such components include (i) a DNA chip, (ii) a device for automatically washing and staining a DNA chip after hybridization, (iii) a scanner for reading fluorescence, and (iv) a device for reading information read. A workstation for processing and analysis; A specific example of such a system is GeneChip from AffyMetrix.^TMAnalysis system. The GeneChip analysis system includes GeneChip as a bioinformatics tool for efficiently using the obtained gene data.^TM Laboratory Information Management System (LIMS^TM) And GeneChip^TM Expression Data Mining Tool (EDMT^TM) Is provided, and the obtained data is output to an SQL-compatible database in a format defined by the Open Consortium for Standardization of Gene-Related Analysis Technology (GATC). It is possible to link. By using the analysis system, data analysis can be performed more efficiently and over a wide range.
[0026]
SEQ ID NO: 1 to SEQ ID NO: 1066 show the nucleotide sequence of a gene whose expression level was found to fluctuate under ischemic conditions in mice according to the present invention, and the amino acid sequence encoded by the gene. Since mice belong to the same mammal as humans, they have high genetic similarity, and have a gene having the nucleotide sequence shown in SEQ ID NOS: 1 to 1066 or a gene encoding the amino acid sequence shown in SEQ ID NOs: 1 to 1066 Genes functionally equivalent to these may also be present in human cells. Therefore, by measuring the expression levels of those human genes, it is possible to perform an ischemic test on a sample derived from human. Here, the “functionally equivalent gene” refers to a gene having the nucleotide sequence shown in SEQ ID NO: 1 to SEQ ID NO: 1066 or a gene encoding the amino acid sequence shown in SEQ ID NO: 1 to SEQ ID NO: 1066, And genes that have homology to these genes and play the same or similar role in vivo in these genes. In addition, information such as the nucleotide sequence and amino acid sequence of a gene present in human cells that is functionally equivalent to a mouse-derived gene can be obtained from a known gene database such as GenBank using a part of the target nucleotide sequence, It can be obtained by a search using a part of the sequence, a gene product name or the like as a keyword.
[0027]
By examining the expression level of the gene whose expression level fluctuates in the ischemic state and further examining the disease level in another disease, the ischemia marker gene whose expression level fluctuates specifically in the ischemic state is identified. It is possible.
[0028]
2. DNA chip for ischemic condition test
A DNA chip in which a part or all of the gene identified in the first section is immobilized on a substrate as a DNA probe can be used as a DNA chip for ischemia test. In particular, as shown in FIG. 2, a DNA chip in which three groups of genes (i.e., a gene having a high expression level, a gene having a medium expression level, and a gene having a low expression level in an ischemic state) are arranged separately. It can be used as a DNA chip capable of evaluating the degree of progress of an ischemic state. There are two types of the DNA chip. One is an adhesive DNA chip manufactured by fixing a DNA probe prepared in advance on a substrate, and the other is a synthetic DNA chip manufactured by synthesizing a DNA probe directly on a substrate. . Here, the “DNA probe” refers to a DNA strand fixed on a DNA chip substrate in order to detect a gene having a DNA strand having a specific base sequence. The method for producing each type of DNA chip will be specifically described below.
[0029]
(1) Production method of a sticking type DNA chip
First, a part or all of the gene whose expression level fluctuates in the ischemic state identified in the above section 1 is prepared by a PCR or a chemical synthesis method for a DNA probe. The DNA probe is placed on the DNA chip substrate so that when the nucleotide chain to be detected having a sequence complementary to the sequence of the probe approaches the DNA probe, hybridization with the nucleotide chain can occur. It must be present as single-stranded DNA. Therefore, in designing a DNA probe, it is preferable to select a sequence so as to minimize the formation of a secondary structure that inhibits hybridization with the nucleotide chain to be detected. Here, the “secondary structure” refers to a stem-loop structure or a hairpin structure that is generated when a DNA probe is folded and a part of the probe hybridizes to another part of the same probe. Whether or not the target sequence forms a secondary structure can be analyzed using commercially available gene analysis software (for example, DNASIS manufactured by Hitachi Software Engineering Co., Ltd.).
[0030]
Preparation of a DNA probe by PCR is performed by using genomic DNA, total RNA, mRNA, and cDNA derived from the test organism as a template in a conventional manner [for example, Sambrook, J et al. , Molecular Cloning: A Laboratory Manual, 2nd Ed. , Cold Spring Harbor Laboratory Press (1989)]. For example, a gene consisting of the nucleotide sequence shown in SEQ ID NO: 1006 can be used as a marker for ischemia test because its expression is significantly increased in an ischemic state. The DNA probe for detecting the gene was derived from an ischemic mouse hippocampus using a sense primer 5′-atgctcttccgagctgttgct-3 ′ (SEQ ID NO: 1067) and an antisense primer 5′-cagctcagttgaacgcctttt-3 ′ (SEQ ID NO: 1068). It can be obtained by PCR using cDNA prepared from mRNA as a template. Whether the amplified fragment obtained by PCR is the target fragment or not is determined by subcloning the amplified fragment into an appropriate vector such as pBlueScript SK (+) (manufactured by STRATAGENE) or pCR2.1 (manufactured by Invitrogen). It can be performed by determining the base sequence. The nucleotide sequence can be determined by a known method such as the Maxam-Gilbert chemical modification method or the dideoxynucleotide chain termination method using M13 phage. Usually, an automatic nucleotide sequencer (for example, 373A DNA manufactured by PERKIN-ELMER) is used. Sequencer or the like).
[0031]
On the other hand, preparation of a DNA probe by a chemical synthesis method can be performed by a normal DNA synthesis method used in the art, such as a phosphoramidite method or a phosphonate method. For example, when synthesizing a DNA probe by the phosphoramidite method, a nucleoside derivative in which a trivalent phosphoramidite residue is introduced into a 3 ′ sugar hydroxyl group is used as a synthesis unit. First, the amidite unit is activated with 1H-tetrazole and reacted with the 5'-terminal hydroxyl group of the DNA chain on the solid phase (step 1) to obtain a trivalent phosphite. Next, the obtained trivalent phosphite is led to a pentavalent phosphate triester through an oxidation reaction (step 2), a capping reaction (step 3), and hydrogenation (step 4). Thereafter, steps 1 to 4 are repeated, and finally the oligomer block having the desired base sequence is cut off from the solid phase and deprotected, whereby the target DNA strand can be obtained.
[0032]
Next, the DNA probe is fixed to a DNA chip substrate. Specific examples of the fixed substrate include a glass plate, a quartz plate, and a silicon wafer. As the size of the substrate, for example, 3.5 mm × 5.5 mm, 18 mm × 18 mm, 22 mm × 75 mm, or the like is used, and this varies depending on the number of spots of the DNA probe on the substrate and the size of the spot. Can be set to As a method for immobilizing DNA, utilizing the charge of DNA, it is electrostatically bound to a solid support surface-treated with a polycation such as polylysine, polyethyleneimine, or polyalkylamine, or an amino group, an aldehyde group, or the like. A DNA probe having a functional group such as an amino group, an aldehyde group, an SH group, or biotin can be covalently bonded to a solid phase surface having a functional group such as an epoxy group.
[0033]
The DNA probe can be spotted on the substrate using an arrayer that can quantitatively spot the DNA probe at a position defined by a size of several tens μm to several hundreds μm. Examples of the spot method include a pin method in which the tip of the pin mechanically contacts the solid phase, an ink-jet method using the principle of an ink jet printer, and a capillary method using a capillary.
[0034]
(2) Method for producing synthetic DNA chip
As a method of synthesizing a DNA probe directly on a substrate, a method of Fodor et al. Combining a photolithography technique and a solid-phase DNA synthesis technique [Fodor, S. et al. P. A. , Et al. , Science, 251: 767-773 (1991)]. That is, first, a synthetic linker having a protecting group that can be removed by a photochemical reaction is bonded onto a substrate, and the substrate is irradiated with light through a blocking substance called a mask, so that only the protecting group in a specific region is eliminated. Let it be. Next, when the substrate is reacted with a nucleotide in which a hydroxyl group is protected, a polymerization reaction occurs only in a portion from which the protecting group has been eliminated. Further, the substrate is irradiated with light using another mask, and the nucleotide polymerization reaction is repeated. In this way, by repeating the coupling reaction with different nucleotide precursors using various masks, a DNA probe having a desired sequence can be synthesized in a specific region on the DNA chip substrate. Oligonucleotides of length Nmer are synthesized in a reaction of N × 4 cycles, and therefore, a DNA probe having a base length of 25 mer can be synthesized in a reaction of 25 × 4 = 100 cycles. The base length of the DNA probe on the DNA chip for ischemia test of the present invention is 10 to 30 mer, preferably 15 to 25 mer.
[0035]
In this type of DNA chip, the specificity of hybridization on the chip may be a problem because the base length of the DNA probe is usually short. This problem can be solved as described below. That is, in order to detect the expression of one specific gene, perfect match (PM) is performed at a plurality of positions (usually more than a dozen) of the target gene (i.e., perfectly complementary). A) An oligonucleotide DNA probe and a mismatch (MM) oligonucleotide DNA probe having a mutation at one base (usually, a base at or near the center) are arranged on the substrate in the same number (FIG. 3). . Subsequently, hybridization is performed on the substrate, and the MM probe is used as an index of the specificity of hybridization. That is, the problem can be solved by calculating the signal ratio between the PM probe and the MM probe and eliminating false positive signals.
[0036]
3. Inspection method for ischemic condition of the present invention
The ischemic condition can be examined by measuring the expression level in a test sample of the gene whose expression level fluctuates in the ischemic condition disclosed in the present invention. Alternatively, the ischemic state can be examined by determining the expression profile of a group of genes including a plurality selected from the genes. Here, “determining the expression profile of the gene group” means that the expression levels of the individual genes constituting the gene group are measured, and the obtained results are summarized in a table or a graph. Specific examples of the ischemic condition include compressional ischemia, obstructive ischemia, spastic ischemia and the like.
[0037]
Examining whether the test sample is in an ischemic state by measuring the expression level in the test sample of the gene whose expression level fluctuates in the ischemic state identified in Section 1 above. Can be. That is, if the expression level of the gene in the test sample fluctuates to the same extent as the fluctuation level in the ischemic state revealed by the present invention, the test sample is evaluated as being in an ischemic state. be able to. For example, a gene having the nucleotide sequence represented by SEQ ID NO: 960 to SEQ ID NO: 1037, SEQ ID NO: 1065, and SEQ ID NO: 1066, or the amino acid sequence represented by SEQ ID NO: 960 to SEQ ID NO: 1037, SEQ ID NO: 1065, and SEQ ID NO: 1066 The encoded gene has a 10-fold or more increase in expression level in ischemic conditions. Therefore, if the expression levels of these genes in the test sample are increased by 10 times or more, the test sample can be evaluated as being in an ischemic state.
[0038]
Further, by measuring the expression level of the gene identified in the above section 1 where little or no change in the expression level is detected in other diseases, that is, the expression level of the ischemic marker gene, the test can be performed more accurately. It can be checked whether the sample is in an ischemic state. That is, if the change in the expression level of the marker gene in the test sample is about the same as the change in the expression level of the gene detected in the present invention, the test sample can be evaluated as being in an ischemic state. .
[0039]
The expression level of the gene can be measured by, for example, dot hybridization, slot hybridization, northern hybridization, or quantitative PCR when the type of the gene to be measured is small. In many cases, it can be measured using a DNA chip.
[0040]
Further, the expression profile of a group of genes including a plurality of genes selected from genes whose expression levels fluctuate in the ischemic state in the test sample is determined in the above section 1, and the obtained profile is compared with the normal profile not in the ischemic state. By comparing with the expression profile of the sample, it is possible to test with higher accuracy whether or not the test sample is in an ischemic state. The measurement of the expression profile of the gene group can be performed more quickly and easily by using a DNA chip. In addition, the expression profile may be classified using cluster analysis described later. As the DNA chip, it is preferable to use a synthetic DNA chip in terms of accuracy, sensitivity, reproducibility, and the like. In addition, an ischemic condition can be examined using the DNA chip of the present invention produced in the second section. For example, as shown in FIG. 2A, the genes are classified into a low expression level gene group, a medium expression level gene group, and a high expression level gene group on a hybridization DNA chip, and the genes belonging to each group are separately placed on the DNA chip. Fixed to. The low expression level gene is a gene in which the transcription level is increased n-fold (where n is 2 or more and less than 5) within 24 hours when the transcription level at 0 hour is 1. An intermediate expression level gene is a gene whose transcription level has increased n-fold (where n is in the range of 5 or more and less than 10). A high expression level gene is a gene whose transcription level is increased by a factor of 10 or more.
[0041]
Using a DNA chip on which a gene whose expression level fluctuates in an ischemic state is fixed, a gene having a base sequence represented by SEQ ID NO: 1 to SEQ ID NO: 1066, or an amino acid sequence represented by SEQ ID NO: 1 to SEQ ID NO: 1066, From the genes to be encoded, for example, 300 or more low expression level genes, 100 to 300 medium expression level genes, and 30 to 100 high expression level genes are selected and fixed. Then, if the expression level of 30 to 100 genes, which are high-expression genes, fluctuates as compared with the gene expression profile derived from a control non-ischemic patient, it is considered to be an initial ischemic state. A decision can be made (FIG. 2B). In addition, when the expression levels of 100 to 300 middle expression level genes as well as the high expression level genes fluctuate, it can be determined that the patient is in a middle-stage ischemic state (FIG. 2C). Furthermore, if the expression levels of at least 300 low-expression genes in addition to the high- and medium-expression genes fluctuate, the ischemic state can be evaluated as late (FIG. 2D).
[0042]
In addition, the fluctuation of the expression level includes a fluctuation toward the increasing side and a fluctuation toward the decreasing side as compared with the normal level. However, the gene having the nucleotide sequence represented by SEQ ID NO: 1 to SEQ ID NO: 1066 or the gene encoding the amino acid sequence represented by SEQ ID NO: 1 to SEQ ID NO: 1066 are all increased in the ischemic state. Therefore, in an ischemic condition test using the fluctuation of the expression level of the gene as an index, only when the expression level increases, the gene whose expression level fluctuates is counted.
[0043]
Furthermore, the expression level of a gene whose expression level does not fluctuate in the ischemic state can be measured and used for the examination of the ischemic state. That is, by measuring the expression level, for a sample evaluated to be ischemic, the expression level is measured for a gene that is shown not to fluctuate in the ischemic state, and the fluctuation in the expression level is observed. If not, the reliability of the evaluation that the test sample is in an ischemic state can be further increased.
[0044]
4. Computer-readable recording medium containing gene expression data in ischemic state and ischemic state identification program, and ischemic state identification program
Expression level data of a gene whose expression level fluctuates in an ischemic state or expression profile data of a group of genes including a plurality of genes selected from the gene is recorded in an appropriate medium, and It can be used as comparison data in data analysis of blood state tests. As a recording medium for recording, any type of recording medium such as a magnetic tape, a CD-ROM, an IC card, and a RAM can be used. Specifically, the expression level of a gene whose expression level fluctuates significantly in an ischemic state is examined in a test sample, and if the expression level is equivalent to the expression level recorded in a recording medium, the test sample (ie, , Test organism) can be evaluated as being in an ischemic state. In addition, comparing the recorded gene expression profile created from the expression level data of each gene of the gene group whose expression level fluctuates in the ischemic state with the gene expression profile of the corresponding gene in the test sample. This enables more accurate evaluation of the ischemic state. That is, in the test sample, if the expression pattern of a plurality of specific genes whose expression levels fluctuate during ischemia is similar to the expression pattern of the relevant gene recorded in the recording medium as described above, the test sample Can be evaluated with a higher probability of being in an ischemic state.
[0045]
A recording medium on which a program for causing a computer to execute the following procedure is recorded is useful as a recording medium including an ischemic state identification program. Here, the ischemic state identification program refers to the stage of the ischemic state (that is, the initial state of ischemia) when the test sample is suspected to be in the ischemic state or is evaluated as the ischemic state. , A middle-stage state or a late-stage state). The program includes (a) a procedure for inputting gene expression level data or gene expression profile data of a test sample, (b) a procedure for recording input data, and (c) recorded data and already recorded. (E) a procedure for comparing the expression level data or gene expression profile data at the time of ischemia, a procedure for determining whether or not the test sample is in an ischemic state based on the comparison results of (d) and (c), and (e) And (c) identifying the clinical stage of the test sample in the ischemic state based on the collation result in the case where the test sample is in the ischemic state. By using a computer in which is installed, the ischemic state can be identified.
[0046]
The ischemic state identification program of the present invention comprises: (a) a means for analyzing the expression level of a gene isolated from a test cell; and (b) an individual test sample using the analysis result obtained in (a) as an index. Means for predicting whether or not is in an ischemic state. The analysis means of the above (a) includes means for detecting the expression level of each gene for a plurality of genes in a certain test cell or test tissue (also referred to as a “detection engine”) and analyzing the obtained detection value. (Also referred to as an “analysis engine”).
[0047]
(1) Gene expression detection engine
In the present invention, gene expression can be detected by digitizing the obtained detection data and using the digital information. The digitization is performed by, for example, digitizing the fluorescence intensity and the like detected on the DNA chip.
[0048]
(2) Analysis engine
The analysis engine is a means for performing analysis processing by multivariate analysis processing such as cluster analysis processing based on data (ie, gene expression level) obtained by the detection engine. The cluster analysis means a method of collecting and classifying “similar objects” for a large number of observation objects (that is, samples) in a field of multivariate analysis by a specific calculation standard (evaluation standard). That is, the cluster analysis refers to a method of simply “classifying” similar samples into the same group for a large number of observed samples.
[0049]
To perform cluster analysis based on the obtained detection data, a “distance matrix” representing the similarity between samples is created. As the distance, a Euclidean distance, a weighted Euclidean distance, a standard Euclidean distance, a Pearson product moment correlation coefficient, and the like are calculated. The concept of these distances is known and can be appropriately selected depending on the purpose of the cluster analysis. Based on the concept of the distance, the distance between the clusters or the distance between the cluster and the individual is calculated, and the clusters are integrated (the two clusters are connected). Methods for integration are known and include, for example, the nearest neighbor method, the farthest neighbor method, the distance between centers of gravity method, the Ward method, and the like.
[0050]
By the above-described classification method, clusters having a “shortest distance” relationship are regarded as “similar” clusters, and are integrated to form a new higher-level cluster. After the cluster of one layer is created, the distance between the clusters is calculated again, a distance matrix is created, the two clusters at the shortest distance are obtained, and the cluster of the next higher layer is created. In this way, a tree diagram (dendrogram) is finally created.
[0051]
Samples in a cluster integrated in a predetermined hierarchy in the tree diagram have some similarity. The samples having the similar relationship can be said to have a certain property in common, and by clarifying the property, the characteristics of the cluster population can be clarified. Therefore, according to this analysis processing, it can be classified into a group of genes having a high expression level and a group of genes having a low expression level. For example, focusing on whether the ischemic state is high or low using the degree of progress of the ischemic state as an index, the ischemic state of a sample belonging to one cluster is high, and the ischemic state of a sample belonging to another cluster is high. The characteristic of low degree can be clarified.
[0052]
Here, a block diagram showing an embodiment of the identification system of the present invention is shown (FIG. 5).
The identification system shown in FIG. 5 includes a CPU 501, a ROM 502, a RAM 503, an input unit 504, a transmission / reception unit 505, an output unit 506, a hard disk drive (HDD) 507, and a CD-ROM drive 508.
[0053]
The CPU 501 controls the entire ischemic condition identification system according to a program stored in the ROM 502, the RAM 503, or the HDD 507, and executes a test process described later. The ROM 502 stores a program or the like for instructing processing necessary for the operation of the identification system. The RAM 503 stores data necessary for executing the inspection processing. The input unit 504 is a keyboard, a mouse, or the like, and is operated when inputting conditions necessary for executing the inspection processing. The transmission / reception unit 505 executes data transmission / reception processing with the database 510 or the like via a communication line based on an instruction from the CPU 501. The output unit 506 performs display processing of various conditions, detected data of an expressed gene, and the like input from the input unit 504 based on a command from the CPU 501. The output unit 506 is exemplified by a computer display or a printer. The HDD 507 stores information on expression patterns of various genes in cells or tissues, reads out stored programs or data based on instructions from the CPU 501, and stores them in, for example, the RAM 503. The CD-ROM drive 508 reads a program or data from the identification program stored in the CD-ROM 509 based on an instruction from the CPU 501 and stores the read program or data in the RAM 503, for example.
[0054]
The CPU 501 supplies data received from the input unit or the like to the output unit 506, and executes prediction based on the data received from the database as to whether or not the test sample is in an ischemic state. The database (DB) is a database in which information on the expression levels (including both absolute and relative levels) of the genes obtained as described above is stored.
[0055]
FIG. 6 is a flowchart illustrating an example when the identification processing is performed by the above-described identification program. The expression pattern of the gene in the test sample is analyzed to identify whether the individual sample is in an ischemic state.
[0056]
In FIG. 6, the identification processing will be described using the cluster analyzer 601 as an example. The cluster analyzer 601 generates a cluster for performing the above-described identification processing. First, gene expression data is input by the external database search input means 602. Until the data input is confirmed, the data input operation is repeated. The information obtained from each tissue or cell by the input of the data is stored in the sample data storage unit 603, provided for cluster analysis, or registered in the database.
[0057]
Next, the data optimizing unit 604 receives the sample data from the sample data storing unit 603 and optimizes the data for cluster analysis. Data optimization is performed by using a method that is most suitable for the sample to be used among methods such as standardization using a median, standardization using a z-score, setting of maximum and minimum values, and logarithmic conversion.
[0058]
The variable list output unit 605 displays a list of variables of sample data on which cluster analysis or the like is performed.
[0059]
Next, the user selects a variable from the variables listed by the variable list output unit 605 by the function of the variable selection unit 606.
[0060]
The selection of a variable by the variable selection means 606 allows one or more specific variables to be freely selected. Normally, there are a large number of candidate variables, so that the user can select any of the variables.
[0061]
When a specific variable is selected by the user, this information is input to the identification sample data file generation means 607 together with the sample data, and the identification sample data file generation means 607 generates a data file of the identification sample.
[0062]
Next, the obtained cluster data file is sent to the identification unit 608, and the identification unit 608 evaluates the degree of cluster separation. An evaluation expression for evaluating the degree of cluster separation can be defined in various forms.
[0063]
The result of the evaluation of the degree of cluster separation by the identification means 608 is passed to the cluster classification means 609. The cluster classification means 609 receives the result of identification by the identification means 608 and refers to the identification conditions set in the identification condition setting means 612 to determine an optimal cluster classification. If the condition for stopping the cluster classification is set, the cluster classification means 609 determines whether to continue or stop the cluster classification. If the condition for continuously stopping cluster classification is not set, the cluster classification means 609 makes the user determine whether to continue or stop cluster classification. When determining to continue the cluster classification, the cluster classification unit 609 outputs the optimal cluster classification obtained in the current process and a signal indicating that the cluster classification is to be continued. The signal indicating that the cluster classification is continued becomes an instruction to return to the processing of the variable list output means 605 after the processing of the tree diagram editing means 611 later.
[0064]
On the other hand, when the cluster classification means 609 decides to stop the cluster classification, it specifies the optimal cluster classification at that stage and outputs a signal to halt the cluster classification. The signal indicating that the cluster classification is to be stopped is an instruction for terminating the cluster classification processing after the processing by the tree diagram editing unit 611.
[0065]
When the processing of the cluster classification means 609 ends, the processing of the tree diagram generation means 610 is started next. The tree diagram generation unit 610 receives the cluster classification determined by the cluster classification unit 609, and displays a tree diagram based on the cluster classification and the attributes of the variables related to each cluster classification. By generating the cluster classification tree diagram by the tree diagram generation unit 610, the user can visually grasp the current state of the cluster classification. The tree diagram generating means 610 displays the gene expression amount based on which the tree diagram was created in accordance with the creation of the tree diagram, so that the user can visually grasp the amount. Next, the tree diagram editing unit 611 allows the user to edit (ie, add, change, or delete) the cluster classification on the display device screen with respect to the cluster classification tree diagram generated by the tree diagram generation unit 610. . The addition, change, or deletion of the cluster classification is performed by the user using the processing command input device on the screen. For example, a predetermined cluster is specified, and a variable of a cluster to be further classified under the specified cluster is specified, a plurality of clusters are integrated, or a branch of the predetermined cluster classification is deleted. The tree diagram editing means 611 provides various tools for supporting the user's editing work on the screen, reads the meaning of the cluster classification editing work by the user, and automatically corrects the data file of each cluster accordingly. . In addition, preferably, the tree diagram editing unit 611 presents a determination to stop the cluster classification by the cluster classification unit 609, and causes the user to input a final determination.
[0066]
As a result, when it is determined to continue the cluster classification repetition processing, the processing is returned to the variable list output unit 605, and the processing from the variable list output unit 605 to the tree diagram editing unit 611 is repeated. It is.
[0067]
From the data analyzed as described above, it is checked which of the ischemic cluster and the normal cluster the test sample is classified into, and it is determined whether or not the test sample is ischemic. can do.
[0068]
5. Screening method for ischemic condition improving drug or ischemic disease therapeutic drug
It is possible to screen for an ischemic condition ameliorating drug or a therapeutic agent for ischemic disease using the expression level of a gene whose expression level increases in an ischemic state, as revealed in the present invention, as an index. That is, by administering a drug to a test animal or a test cell, (a) whether or not the expression level of the above gene returns to the expression level in a normal tissue, or (b) expression of a group of genes including a plurality of the genes. Determine whether the profile reverts to a normal expression profile in normal tissues. If the expression level of the gene returns to the expression level in normal tissues as a result of administering a certain drug, the drug can be evaluated as a candidate substance for an ischemic condition improving drug or a therapeutic drug for ischemic disease.
[0069]
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, the present invention will be described specifically with reference to Examples, but the scope of the present invention is not limited thereto.
[0070]
[Example 1] {Identification of gene expressed during ischemic condition by DNA chip}
GeneChip System^TM(Affymetrix) was used to identify genes that are expressed in the ischemic state by the procedure shown in FIG. That is, first, bcl BLACK mice of 8 to 10 weeks of age were anesthetized with inflation, the bilateral carotid arteries were exposed and ligated for 20 minutes to cut off the blood flow and put the mice into an ischemic state. Thereafter, the blood flow was restored, and at 0, 2, 6, 12, and 24 hours after the blood flow was restored, the mice were sacrificed, the hippocampus was removed from both sides, and immediately cryopreserved. Next, about 2 μg of poly (A) + mRNA was extracted from 1 g of the frozen sample. Next, cDNA was synthesized using reverse transcriptase. Furthermore, biotin-labeled cRNA was prepared from this cDNA by in vitro transcription. Labeled cRNA with Mg²⁺Heat treatment in the presence fragmented to a size of about 50 mer. After labeling the internal standard, it was added to the sample, and this sample was added to the GeneChip^TM Mu6500 (manufactured by AffiyMetrix) was injected. After hybridization in an oven, the chip was washed in a Fluidic station. The chip information was captured with a GeneArray scanner. The obtained data was processed and analyzed using a bioinformatics system. The results are shown in Tables 1 to 3. Table 1 shows genes whose transcription level increased n-fold (n is in the range of 2 or more and less than 5) within 24 hours, when the transcription level at 0 hour is 1. Similarly, Table 2 shows genes whose transcription level was increased n-fold (n is in the range of 5 or more and less than 10). Table 3 shows the genes whose transcription levels increased by a factor of 10 or more. The accession number (ACCESSION NO.) Is the accession number of GenBank.
[0071]
[Table 1]

[0072]
[Table 2]

[0073]
[Table 3]

[0074]
All documents, patents and patent applications cited herein are hereby incorporated by reference in their entirety.
[0075]
Industrial applicability
By analyzing gene expression in a test sample, an ischemic condition is examined. Such analysis is expected to be applied to the prevention and treatment of ischemia. In addition, a novel method for screening for ischemic prophylactic and therapeutic agents using the expression level of a gene specifically expressed in the ischemic state as an index is provided.
[0076]
Sequence listing free text
SEQ ID NO: 1067: Synthetic DNA
SEQ ID NO: 1068: Synthetic DNA

[Brief description of the drawings]
FIG.
FIG. 1 is a diagram showing a procedure for identifying a gene whose expression level fluctuates in an ischemic state using a DNA chip.
FIG. 2
FIG. 2 is a diagram showing one embodiment of the DNA chip for ischemic condition inspection of the present invention and the results expected when a sample derived from a patient to be tested is hybridized using the DNA chip.
FIG. 3
FIG. 3 is a diagram showing one embodiment of a synthetic DNA chip and a procedure for detecting a gene in a test sample using the DNA chip.
FIG. 4
FIG. 4 shows the GeneChip^TMFIG. 5 is a diagram showing a procedure for measuring a gene expression level by using FIG.
FIG. 5
FIG. 5 is a block diagram of the ischemic condition identification system.
FIG. 6
FIG. 6 is a flowchart showing an example of processing by the ischemic state identification program.
[Explanation of symbols]
501: CPU, 502: ROM, 503: RAM, 504: input unit, 505: transmission / reception unit, 506: output unit, 507: HDD, 508: CD-ROM drive, 509: CD-ROM, 510: database 601 : Cluster analyzer, # 602: external database search and input means, # 603: sample data storage means, $ 604: data optimization means, $ 605: variable list output means, $ 606: variable selection means, $ 607: evaluation sample data file generation means 608: evaluation means, # 609: cluster classification means, # 610: tree diagram generation means, # 611: tree diagram editing means, # 612: evaluation condition setting means

Claims (16)

Examining the ischemic state by measuring the expression level of a specific gene in the test sample or determining the expression profile of a group of genes including a plurality of genes selected from the specific gene in the sample; Inspection method for ischemic condition. The specific gene is
(A) a gene having any of the nucleotide sequences shown in SEQ ID NOs: 1 to 1066, or a gene encoding any of the amino acid sequences shown in SEQ ID NOs: 1 to 1066, or (b) any of the above nucleotide sequences A gene functionally equivalent to a gene having the above, or a gene functionally equivalent to a gene encoding any of the above amino acid sequences,
The method of claim 1, wherein The method according to claim 1 or 2, wherein the measurement of the expression level or the determination of the expression profile is performed on a DNA chip. 4. The method according to claim 3, wherein the DNA chip is a synthetic DNA chip. The method according to any one of claims 1 to 4, wherein the ischemic condition is at least one selected from the group consisting of compressive ischemia, obstructive ischemia and spastic ischemia. The following genes (a) or (b):
(A) a gene having any of the nucleotide sequences shown in SEQ ID NOs: 1 to 1066, or a gene encoding any of the amino acid sequences shown in SEQ ID NOs: 1 to 1066, or (b) any of the above nucleotide sequences A gene functionally equivalent to a gene having the above, or a gene functionally equivalent to a gene encoding any of the above amino acid sequences,
DNA chip for ischemic condition inspection, wherein a part or all of the DNA chip is immobilized on the surface. The DNA chip according to claim 6, wherein the ischemic condition is at least one selected from the group consisting of compressional ischemia, obstructive ischemia, and spastic ischemia. A method for screening an ischemic condition improving drug or a therapeutic agent for ischemic disease, by administering a drug to a test animal or test cells,
(A) whether or not the expression level of a specific gene whose expression level fluctuates in an ischemic state returns to a normal expression level; or (b) the expression profile of a gene group containing a plurality of the specific gene is normal Whether to return to the expression profile,
The method as described above, comprising selecting a candidate drug using as an index, and returning to a normal expression level or a normal expression profile indicates that the drug is a candidate drug. A specific gene whose expression level fluctuates in an ischemic state is
(A) a gene having any of the nucleotide sequences shown in SEQ ID NOs: 1 to 1066, or a gene encoding any of the amino acid sequences shown in SEQ ID NOs: 1 to 1066, or (b) any of the above nucleotide sequences A gene functionally equivalent to a gene having the above, or a gene functionally equivalent to a gene encoding any of the above amino acid sequences,
9. The method according to claim 8, wherein The method according to claim 8 or 9, wherein the ischemic condition is at least one selected from the group consisting of compressive ischemia, obstructive ischemia and spastic ischemia. The following data of (a) or (b):
(A) expression level data of a gene whose expression level fluctuates in an ischemic state, or (b) expression profile data of a gene group including a plurality of genes selected from the gene,
A computer-readable recording medium on which is recorded. A gene whose expression level fluctuates in an ischemic state is
(A) a gene having any of the nucleotide sequences shown in SEQ ID NOs: 1 to 1066, or a gene encoding any of the amino acid sequences shown in SEQ ID NOs: 1 to 1066, or (b) any of the above nucleotide sequences A gene functionally equivalent to a gene having the above, or a gene functionally equivalent to a gene encoding any of the above amino acid sequences,
The recording medium according to claim 11, which is: 13. The recording medium according to claim 11, wherein the ischemic condition is at least one selected from the group consisting of compressive ischemia, obstructive ischemia, and spastic ischemia. The following steps:
(A) inputting expression level data or expression profile data of a specific gene in a test sample,
(B) recording the input data,
(C) comparing the recorded data with the already recorded expression level data or expression profile data of the gene in the ischemic state,
(D) a procedure for determining whether or not the test sample is in an ischemic state based on the verification result in (c); and (e) verification in (c) when the test sample is in an ischemic state. A procedure for identifying the clinical stage of the ischemic state of the test sample based on the result,
A computer-readable recording medium on which a program for causing a computer to execute is recorded. The gene has the following (a) or (b):
(A) a gene having any of the nucleotide sequences shown in SEQ ID NOs: 1 to 1066, or a gene encoding any of the amino acid sequences shown in SEQ ID NOs: 1 to 1066, or (b) any of the above nucleotide sequences A gene functionally equivalent to a gene having the above, or a gene functionally equivalent to a gene encoding any of the above amino acid sequences,
The recording medium according to claim 14, which is: 16. The recording medium according to claim 14, wherein the ischemic condition is at least one selected from the group consisting of compressional ischemia, obstructive ischemia, and spastic ischemia.

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