JP2009036681A - Method for measuring activity of fibroblast growth factor receptor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for accurately and quantitatively measuring the activity of FGFRs (fibroblast growth factor receptors). <P>SOLUTION: The method for quantitatively measuring the activity of the fibroblast growth factor receptors includes steps for developing the fibroblast growth factor receptors in cells, activating the developed fibroblast growth factor receptors, acquiring an image which shows the distribution of the activated receptors in the cells, and analyzing the acquired image. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a method for measuring the activity of a fibroblast growth factor receptor (FGFR). More specifically, the present invention relates to a method for quantitatively measuring the activation state of FGFR using an image analyzer.

  Fibroblast growth factor receptor (FGFR) is known to have an important role in various physiological functions such as cell proliferation, differentiation, and neurotransmission. Currently, various methods have been developed to measure the activation state of FGFR present on the cell surface. For example, Non-Patent Document 1 discloses that a certain type of fibroblast growth factor (FGF) quantifies its activation state by inducing differentiation of nerve cells. Non-Patent Document 2 discloses that a lymphocyte cell line cultured cell line BaF is used to measure a novel amount of DNA synthesis or an increase in the number of cells, and to measure DNA synthesis promoting activity and cell proliferation activity of FGF. Yes. In addition, FGFR has been reported to be localized to the cell nucleus (Non-Patent Document 3) or intracellular vesicles (Non-Patent Document 4) upon activation. Is labeled and observed under a microscope.

  However, since the conventional method is greatly influenced by the endogenous FGFR expression of cells, there is a problem that only a few cell lines can be used for measurement. In particular, many adherent cultured cells are not suitable for this measurement. Further, the conventional method is complicated in operation, requires a skillful experimental technique, and further requires a large amount of material cells. Furthermore, since many intermediate experiments are performed before results are obtained, it is not suitable for processing a large number of specimens and is not suitable for quantitative measurement.

In Non-Patent Documents 5 and 6, in order to measure the activity of a G protein-coupled receptor molecule, the distribution of granular structures formed by the receptor is continuously imaged and analyzed using a specific algorithm. It is disclosed. G protein-coupled receptor molecules are known to migrate to small structures such as intracellular vesicles as an activation trigger and form a granular pattern, but they are a group different from FGFR. No cell surface receptor belonging to the above, and no example of FGFR measured by such a method has been reported.
J. Biol. Chem. 273: 29262-29271 (1998) J. Biol. Chem. 277: 42815-42820 (2002) J. Biol. Chem. 16: 14-23 (2005) J. Cell. Science. 111: 3517-3527 (1998) Cytometry A 65: 69-76 (2005) J. Biomol. Screen 10: 476-484 (2005)

  An object of the present invention is to provide a method for quantitatively measuring FGFR activity easily and accurately. More specifically, the present invention relates to a method for quantitatively measuring the activity of FGFR by imaging granular structures formed by activated FGFR in cells and analyzing them using an image analyzer. provide. Still another object of the present invention is to provide a method for evaluating the degree of physical or chemical action given to cells.

  According to the present invention, there is provided a method for quantitatively measuring the activity of a fibroblast growth factor receptor, the step of expressing the fibroblast growth factor receptor in a cell, and the expressed fibroblast growth factor receptor. There is provided a method comprising a step of activating a body, a step of obtaining an image representing a distribution of the activated receptor in the cell, and a step of analyzing the obtained image.

  Here, in the step of expressing the fibroblast growth factor receptor in the cell, a gene encoding the receptor is preferably introduced into the cell by a gene recombination technique. In addition, the receptor is preferably activated by bringing the fibroblast growth factor receptor into contact with a substance that activates the receptor. In addition, the acquisition of the image is preferably performed by labeling the receptor with immuno-antibody staining or fluorescent labeling and taking a microscopic image.

  In one embodiment, the step of analyzing the obtained image includes a step of recognizing a granular structure existing in the cell, and the granular structure is converted into roundness, aggregation degree, fluorescence intensity and area. And a step of calculating a quantity per cell of the granular structure having a desired property among the classified granular structures.

  Another aspect of the present invention is a method for screening a substance that regulates the function of a fibroblast growth factor receptor, the step of expressing the fibroblast growth factor receptor in a cell, and the expression of the receptor There is provided a method comprising a step of bringing a test substance into contact with a test cell, a step of obtaining an image representing the distribution of the receptor in the cell, and a step of analyzing the obtained image. Still further, a method comprising the step of quantitatively estimating the titer of the substance by quantifying the activity of the receptor based on the distribution of the fibroblast growth factor receptor in the cell. Provided.

  ADVANTAGE OF THE INVENTION According to this invention, the method of measuring the activity of a fibroblast growth factor receptor simply and accurately can be provided using arbitrary adherent cells, and in particular, a desired subtype of fibroblast proliferation. It is possible to selectively measure factor receptors. Moreover, the screening method of the active substance with respect to this receptor can be provided.

  The receptor for fibroblast growth factor (FGF), when activated, induces neuronal differentiation and promotes cell proliferation. Therefore, conventionally, a method for evaluating the activation state of a receptor by counting the number of proliferated cells has been generally performed.

  In contrast, the present invention provides a method for quantifying the activation state of fibroblast growth factor receptor (FGFR) easily and accurately by using a microscopic image analysis system. Moreover, according to the method of the present invention, it is possible to easily screen for substances that regulate the function of FGFR.

  In general, receptor molecules are present on the surface of cells, but when they react with factors, the receptor molecules gather and embed, become granular below the cell surface, and eventually enter the cell. In the present specification, this is referred to as a granular structure. When activated, FGFR accumulates in cytoplasmic vesicles and shows a granular distribution within the cell.

  In the measurement method of the present invention, first, fibroblast growth factor receptor (FGFR) is expressed in cells. The method for expressing FGFR in cells can be performed by gene recombination. Specifically, an expression plasmid vector for expressing FGFR cDNA is constructed, and this plasmid vector is introduced into cells. Construction of the plasmid vector, introduction into the cell, and expression in the cell can be performed by methods well known in the art.

  Next, FGFR expressed on the cell surface is activated. The activation may be performed by adding a substance that activates FGFR into the cell culture medium, or may be performed by changing physical conditions such as temperature. After activating FGFR, the cells are fixed with a fixative.

  When FGFR is activated, it forms a granular structure as described above. Therefore, the activity of FGFR can be estimated by observing the distribution of FGFR in the cell. Therefore, in this invention, the image showing distribution in the cell of FGFR is acquired. Image acquisition may be performed by any method, but it is particularly preferable to acquire an electronic image using a microscope. For imaging, a commonly used CCD camera or the like can be used, but is not limited thereto.

  FGFR expressed in cultured cells can be stained or fluorescently labeled by a fluorescent antibody method, an enzyme antibody method, etc., and can be detected with a microscope. For example, a fusion gene of a transcriptional regulatory unit of a gene encoding FGFR and a reporter gene such as a fluorescent or luminescent protein is introduced into a cell in advance by gene recombination technology, and the expression of the fusion gene is detected. Also good. Alternatively, a specific amino acid sequence called a tag sequence may be inserted into the FGFR gene. The amino acid tag sequence appears in a part of the receptor, and by reacting a labeled antibody that recognizes the tag sequence with this amino acid tag sequence, the receptor molecule can be detected. According to the genetic recombination technique as described above, a specific amino acid sequence of the FGFR molecule can be identified, and thereby, a desired subtype receptor can be selectively detected. Alternatively, FGFR expressed in cultured cells can be detected by imaging a transmitted light image, a differential interference image, an optical phase difference image, and the like.

  Next, the obtained image is analyzed, and the distribution state of FGFR is analyzed. In this analysis, the granular structure existing in the cell is recognized. Then, the recognized granular structure is classified according to at least one property selected from, for example, roundness, aggregation, fluorescence intensity, and area. Among the classified granular structures, the number of granular structures having desired properties per cell is counted. For example, a granular structure having a value larger than a preset threshold value may be counted. Moreover, you may count what has a certain predetermined value in a certain range.

  In one embodiment, the analysis of the microscopic image of FGFR is characterized by classifying granular structures formed by FGFR according to their size and measuring the number of granular structures of a specific size per cell. To do. Alternatively, the structure of maximum brightness of each cell is measured. This method is suitable for automation of measurement because the measurement is simplified and the throughput is improved.

  Each step in the method of the present invention described above can be performed using an apparatus capable of acquiring and analyzing cell images. For example, a system in which a microscope imaging device and an image analysis device including image analysis software are integrated can be used. In particular, a system that continuously and automatically acquires a microscope image and analyzes an image is preferably used.

  The above method may use any image acquisition device and analysis software. For example, the main product of (CompuCyte, Cambridge, MA, USA) is a microscope-based cytometer (Laser Scanning Cytometer, LSC (registered trademark)) )) An imaging cytometer iCyte (trademark) designed based on the series (2, 3) is preferably used.

  However, since the shape of the granular structure of FGFR is not clearer than that of a normal receptor molecule, automatic recognition by an image analyzer is difficult. Therefore, in the present invention, the setting value of the microscope image analysis system is changed to improve the recognition of the granular structure formed by the FGFR. Specifically, since the granular structure of FGFR is relatively fine, the area threshold for image recognition is set to be small, and the fine structure can be captured.

  An example of an analysis algorithm for measuring a granular structure using such a system will be described. First, it recognizes all the granular structures present in the cells. As described above, in the granular structure, for example, the molecules constituting the granular structure are labeled with a fluorescent dye or a biological (or chemical) luminescent dye, and the biological (chemical) luminescence / fluorescence from the label is obtained. This can be done by detecting. For the labeling, any method known to those skilled in the art may be used. For example, the labeling can be performed using a binder such as an antibody that recognizes a molecule constituting the granular structure. In this case, the antibody may be labeled with a fluorescent substance such as FITC or bioluminescence such as luciferase directly with the antibody, or may be indirectly labeled with a labeled secondary antibody. . Alternatively, any molecule to which a luminescent or fluorescent protein such as a bioluminescent fusion protein or a GFP fusion protein is bound in advance may be expressed in the cell, and luminescence due to bioluminescence / fluorescence may be detected.

  When fluorescence-labeled cells are irradiated with appropriate excitation light, intrinsic fluorescence is produced. For example, when labeled with GFP, excitation is performed with an argon ion laser (488 nm), and fluorescence at 515 to 545 nm is detected. An image of the detected fluorescence is acquired, and by setting a certain fluorescence intensity threshold, a map contour line is drawn on the image, and local aggregation of an arbitrary molecule is recognized as a granular structure. At this time, the threshold value is set so as to reflect the position and form of the cell nucleus (structure outline). These recognized granular structures are regarded as independent events, and their properties (roundness, aggregation, fluorescence intensity, area, etc.) are automatically measured using an image analyzer and statistically analyzed. To do. In addition, when labeling with a luminescent substance, it is not necessary to irradiate excitation light.

  Next, the structure is classified based on, for example, the area of the existing structure outline. Moreover, those skilled in the art can determine the desired size of the area, that is, the size of the granular structure, based on various experiments and experiences. For example, for each assay system, the number of granular structures of various sizes per cell may be calculated by the method of the present invention, and the granular structures having a size reflecting a desired cell state may be selected. Thus, by classifying according to the size of the granular structure in the cell, the correlation between the generation of the granular structure and the cell state can be more reflected, and the cell state can be measured more accurately. it can.

  In general, a positive correlation is observed between the increase in the activation state of FGFR and the degree of generation of granular structures. For example, the number of granular structures per cell can be expressed as the activation state of FGFR. Therefore, it is possible to estimate the active state of FGFR using the numerical value obtained by the above analysis as an index.

  Any cell may be used as the FGFR-expressing cell used in the method of the present invention. For example, cultured cells can be used, and cells derived from blood such as peripheral venous blood, organs, and tissues may be used. The cells may be derived from any species, and may be humans, non-human animals and plants, and microorganisms such as viruses, bacteria, bacteria, yeasts, and mycoplasmas. In particular, any adherent cell can be used by measuring using a microscope image analysis system in accordance with the present invention. Adhesive cells tend to form a monolayer and are flatly attached to observation supports such as glass slides, petri dishes, and microplates, and the cells are immobilized, making them particularly suitable for detection with a microscope. Yes. However, many adherent cells inherently express FGFR, and may have many types of receptors other than FGFR on the surface, which cannot be distinguished from the FGFR to be detected. The cells that can be used in the detection test were limited. However, according to the method of the present invention, since any desired subtype of FGFR can be specifically detected, any adherent cell can be used.

  As described above in detail, according to the method of the present invention, a receptor subtype is specifically identified by applying stimulation to a cell surface activated only with a desired FGFR by a drug or the like to be examined. Therefore, it is possible to selectively label and observe only the receptor of the desired subtype to be investigated. In addition, since the receptor that has not been activated by reacting with the compound does not become granular, according to the method of the present invention, only the activated receptor can be detected, and simple and highly accurate detection is possible. It is.

  In another embodiment of the present invention, a substance that regulates the function of FGFR can be screened by measuring the activity of FGFR. A test substance is brought into contact with the cell, and the granular structure in the cell is measured and analyzed according to the method of the present invention. When FGFR is activated to form a granular structure by contact with a test substance, the test substance is considered to be an agent for FGFR. Thus, the method of the present invention can be used to screen whether the test substance is an FGFR agonist. Furthermore, the titer of the substance can be quantitatively estimated by quantifying the activity of the receptor based on the distribution of FGFR in the cell.

  In still another embodiment of the present invention, a substance that regulates the function due to the interaction between FGFR and its ligand can be screened. First, a test substance is brought into contact with cells. Next, a ligand is administered to the cells contacted with the test substance. Next, the granular structure formed by FGFR by the method of the present invention is measured. If the granular structure is reduced by contact with the test substance, the test substance is considered to be an antagonist of cell function via the FGFR / ligand. Thus, it can be accurately measured whether or not the test substance affects the interaction between FGFR and its ligand.

  Fibroblast growth factor receptor (FGFR) cDNA was obtained by RT-PCR (using Quiagen RT-PCR kit) using mouse ICR fetal total RNA as a template. The base sequence of the mouse FGFR cDNA was obtained from the GenBank database with the accession number NM_010207, and a primer DNA for reverse transcription PCR was synthesized based on this base sequence. Subsequently, an expression plasmid DNA in which this cDNA was inserted into an animal cell expression vector pSI (Promega) was prepared.

  Cultured cells (CHO cells) were cultured by a usual method using McCoy's 5A medium, and the plasmid DNA was introduced into the cells using Llpofectamine 2000 (Invltrogen). After continuous culture, the cells were cultured in a serum-free medium for 16 hours to make the FGFR on the cells inactive.

  Fibroblast growth factor (FGF) produced in E. coli (aFGF, Peprotech) was diluted stepwise, added to the CHO cells, and incubated at 37 ° C. for 30 minutes. Subsequently, cells were fixed by adding 2% paraformaldehyde. An antibody against FGFR (anti-Bek antibody, SanteCruz) was bound to the fixed cells as a primary antibody, and excess antibody was washed. Subsequently, Alexa488-labeled anti-rabbit IgG antibody (Invitrogen) was bound to visualize the presence of FGFR. After washing away excess antibody, the cell nucleus was stained with DAPI fluorescent dye, and at the same time, RNA in the cell was digested with RNase.

  The cell specimen produced as described above was imaged and analyzed by a system in which microscope image acquisition / analysis was integrated (see Non-Patent Document 5). FIG. 1 shows the result of analyzing the CHO cell introduced with the FGFR2 IIIc expression vector by the above procedure. FIG. 1 shows the result of quantifying the degree of FGFR aggregation by image analysis. It has been shown that relative activity increases as the concentration of FGFR added in the cell culture increases. Therefore, it was shown that the activation state of FGFR can be estimated by the measurement method according to the present invention.

The graph which digitized the aggregation degree of the fibroblast growth factor receptor with the automation system.

Claims (7)

A method for quantitatively measuring fibroblast growth factor receptor activity comprising:
Expressing a fibroblast growth factor receptor intracellularly;
Activating the expressed fibroblast growth factor receptor;
Obtaining an image representative of the distribution of the activated receptor in the cell;
Analyzing the obtained image;
A method comprising:   The method according to claim 1, wherein the step of expressing the fibroblast growth factor receptor in the cell comprises introducing a gene encoding the receptor into the cell by a gene recombination technique.   The method according to claim 1 or 2, wherein the receptor is activated by bringing the fibroblast growth factor receptor into contact with a substance that activates the receptor.   The method according to any one of claims 1 to 3, wherein the acquisition of the image is performed by labeling the receptor with immuno-antibody staining or fluorescent labeling and capturing a microscopic image. The step of analyzing the obtained image includes:
Recognizing granular structures present in the cells;
Classifying the granular structure according to at least one property selected from roundness, aggregation, fluorescence intensity, and area;
The method as described in any one of Claims 1-4 including the process of calculating the quantity per cell of the granular structure which has a desired property among the classified granular structures. A method for screening a substance that regulates the function of a fibroblast growth factor receptor,
Expressing a fibroblast growth factor receptor intracellularly;
Contacting a test substance with a cell in which the receptor is expressed;
Obtaining an image representing the distribution of the receptor in the cell;
Analyzing the obtained image;
A method comprising:   The method further comprises the step of quantitatively estimating the titer of the substance by quantifying the activity of the receptor based on the distribution of the fibroblast growth factor receptor in the cell. 6. The method according to 6.

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