WO2001088188A2

WO2001088188A2 - Method for examining ischemic conditions

Info

Publication number: WO2001088188A2
Application number: PCT/JP2001/004192
Authority: WO
Inventors: Koichi Ishikawa; Satoshi Asai; Yasuo Takahashi; Toshihito Nagata; Yukimoto Ishii
Original assignee: Nihon University, School Juridical Person
Priority date: 2000-05-18
Filing date: 2001-05-18
Publication date: 2001-11-22
Also published as: JP2004512014A; WO2001088188A3; AU2001256780A1

Abstract

The present invention provides a method for examining ischemic conditions, comprising measuring the expression levels of particular genes in a test sample or determining the expression profile of a gene group in the sample comprising a plurality of genes selected from said particular genes.

Description

DESCRIPTION

METHOD FOR EXAMINING ISCHEMIC CONDITIONS

TEHCHNICAL FIELD

The present invention relates to a method for examining ischemic conditions by measuring the expression levels of particular genes in a test sample or by determining the expression profile of a gene group in the sample comprising a plurality of genes selected from the particular genes.

BACKGROUND ART

Cancer, cerebral apoplexy and heart diseases are called three major adult diseases and they occupy about 60% of the causes of death in the Japanese. Of these, cerebral apoplexy and heart diseases are often caused by ischemia. Thus, early detection of ischemic conditions makes it possible to prevent these diseases from occurring. Ischemia is local anemia and may be classified into groups such as compressive ischemia caused by constriction or occlusion in arterial walls due to external pressure from tumor or the like; occlusive ischemia caused by changes inside the blood vessels or in the blood vessels themselves such as thrombosis or arterial sclerosis; and vasospastic ischemia caused by vasospasms such as cerebral anemia or angina, from the viewpoint of the mechanism of its occurrence. Ischemia in the brain triggers ischemic cerebral apoplexy such as cerebral infarction, and ischemia in the heart triggers ischemic heart diseases such as myocardial infarction. Thus, for the prevention of these diseases, it is important, first of all, to find ischemic conditions as early as possible and to receive appropriate treatment.

As a method for examining ischemic conditions, a method in which abnormality in the cardiac wall movement is used as an indicator (e.g., quantitative analysis of ventricular forms/ultrasonic images, or detection of decrease in tissue systole speed); a method in which abnormality in hemodynamics is used as an indicator (e.g., analysis of the pattern of blood flow rate into the left ventricle, or nuclear medicine examination) and the like have been known to date. Among all, nuclear medicine examination is a method which can examine ischemic conditions accurately. However, this method is disadvantageous to subjects because it involves exposure to radiation, requires a long time for examination, and is expensive. Under circumstances, a simple examination method for ischemic conditions has been desired which imposes less burden to subjects and can be carried out routinely as a part of health examination.

DISCLOSURE OF THE INVENTION

It is an object of the present invention to provide a method for examining ischemic conditions simply by measuring the expression levels of particular genes in a test sample or by determining the expression profile of a gene group in the sample comprising a plurality of genes selected from the particular genes.

As a result of intensive and extensive researches toward the solution of the above problems, the present inventors have succeeded in identifying those genes expressed under ischemic conditions and in elucidating the expression profile of the genes. Thus, the present invention has been achieved.

The present invention relates to a method for examining ischemic conditions, comprising measuring the expression levels of particular genes in a test sample or determining the expression profile of a gene group in the sample comprising a plurality of genes selected from the particular genes. Specific examples of the particular genes include (a) genes having any of the nucleotide sequences shown in SEQ JJD NO: 1 through SEQ ID NO: 1066 or genes encoding any of the amino acid sequences shown in SEQ ID NO: 1 through SEQ ID NO: 1066; or (b) genes functionally equal to the genes having any of the above-described nucleotide sequences or genes functionally equal to the genes encoding any of the above-described amino acid sequences. The measurement of expression levels and the determination of expression profile may be carried out using a DNA chip (e.g., a synthetic-type DNA chip). Specific examples of the ischemic conditions include compressive ischemia, occlusive ischemia and vasospastic ischemia.

Further, the present invention relates to a DNA chip for examining ischemic conditions, which carries a part or all of the following genes (a) or (b) immobilized on its surface: (a) genes having any of the nucleotide sequences shown in SEQ JJD NO: 1 through SEQ ID NO: 1066 or genes encoding any of the amino acid sequences shown in SEQ ID NO: 1 through SEQ ID NO: 1066; or (b) genes functionally equal to the genes having any of the above-described nucleotide sequences or genes functionally equal to the genes encoding any of the above-described amino acid sequences. Specific examples of the ischemic conditions include compressive ischemia, occlusive ischemia and vasospastic ischemia.

Further, the present invention relates to a method of screening for ischemic condition- improving drugs or therapeutics for ischemic diseases. This method is characterized by selecting candidate drugs using as an indicator whether or not:

(a) the expression levels of particular genes of which expression levels change under ischemic conditions return to a normal expression levels; or

(b) the expression profile of a gene group comprising a plurality of the particular genes returns to a normal expression profile; by the administration of a drug to a test animal or test cell, wherein the returning to the normal expression levels or normal expression profile indicates that the drug is a candidate drug. Specific examples of the particular genes include (a) genes having any of the nucleotide sequences shown in SEQ ID NO: 1 through SEQ ED NO: 1066 or genes encoding any of the amino acid sequences shown in SEQ ID NO: 1 through SEQ ED NO: 1066; or (b) genes functionally equal to the genes having any of the above-described nucleotide sequences or genes functionally equal to the genes encoding any of the above- described amino acid sequences. Specific examples of the ischemic conditions include compressive ischemia, occlusive ischemia and vasospastic ischemia.

Further, the present invention relates to a computer-readable record medium in which the following data (i) or (ii) have been recorded: (i) expression level data of particular genes of which expression levels change under ischemic conditions, or (ii) expression profile data of a gene group comprising a plurality of genes selected from the particular genes. Specific examples of the particular genes include (a) genes having any of the nucleotide sequences shown in SEQ ED NO: 1 through SEQ ID NO: 1066 or genes encoding any of the amino acid sequences shown in SEQ ED NO: 1 through SEQ ED NO: 1066; or (b) genes functionally equal to the genes having any of the above-described nucleotide sequences or genes functionally equal to the genes encoding any of the above- described amino acid sequences. Specific examples of the ischemic conditions include compressive ischemia, occlusive ischemia and vasospastic ischemia.

Further, the present invention relates to a computer-readable record medium in which a program that directs a computer to execute the following procedures has been recorded:

(a) procedures to input expression level data or expression profile data of particular genes in a test sample;

(b) procedures to record the input data;

(c) procedures to check the recorded data with already recorded expression level data or expression profile data of the particular genes under ischemic conditions;

(d) procedures to determine whether the test sample is under ischemic conditions or not based on the checking results obtained in (c); and

(e) if the test sample has been determined as being under ischemic conditions, procedures to identify the clinical stage of the ischemic conditions of the test sample based on the checking results obtained in (c). Specific examples of the particular genes include (a) genes having any of the nucleotide sequences shown in SEQ ED NO: 1 through SEQ ED NO: 1066 or genes encoding any of the amino acid sequences shown in SEQ ED NO: 1 through SEQ ED NO: 1066; or (b) genes functionally equal to the genes having any of the above-described nucleotide sequences or genes functionally equal to the genes encoding any of the above-described amino acid sequences. Specific examples of the ischemic conditions include compressive ischemia, occlusive ischemia and vasospastic ischemia.

Hereinbelow, the present invention will be described in detail.

The present specification encompasses the contents of the specification and drawings of Japanese Patent Application No. 2000-145977 based on which the present application claims priority.

The present invention relates to a unique method for examining ischemic conditions using, as an indicator, expression levels of particular genes or an expression profile of a particular gene group. The term "expression level" used herein refers to an absolute or relative amount of the transcript (i.e., mRNA) of a particular gene; or an absolute or relative amount of the translation product (i.e., protein) of a particular gene. The term "expression profile" used herein refers to expression levels of a plurality of genes collected and arranged in tables, graphs, or the like.

1. Identification of Genes of which Expression Levels Change under Ischemic Conditions

Genes of which expression levels change under ischemic conditions may be identified by, for example, the differential RNA display method [Liang, P. et al., Science 257:967-971 (1992)], the hybrid subtraction method, or a method using a DNA chip. For example, a method of identifying the above genes using a DNA chip may be carried out as illustrated in Fig. 1. Briefly, a plurality of pieces of DNA information (cDNA/EST/oligoDNA collection) are obtained from a DNA database where genomic sequences, cDNA sequences or EST sequences have been recorded. Then, a wide variety of genes of known sequences are immobilized on a DNA chip. Subsequently, labeled DNA or RNA which has been prepared from mRNA derived from biosamples under ischemic conditions is hybridized with the DNA chip. The hybridization strength at each spot of the resultant hybridization pattern is then measured to thereby measure the expression level of each gene. Thus, an expression profile is obtained. Hereinbelow, a method using a DNA chip will be described in more detail.

(1 ) Preparation of Poly(A)+mRNA from Test Samples

First, poly(A)+mRNA must be prepared from test samples such as tissue or cell to examine expression levels of particular genes in the samples using a DNA chip. Specific examples of test samples useful for the preparation of poly(A)+mRNA to be used in the identification of genes of which expression levels change under ischemic conditions include biotissues (e.g., blood tissue, brain tissue, heart tissue or renal tissue) derived from experiment animals (e.g., mice, rats, guinea pigs, rabbits, dogs, cats, pigs or cows) in which ischemic conditions have been induced artificially or derived from humans under ischemic conditions. It is said that about 80% of genes which may be expressed in a living body are being expressed in the brain. Thus, by examining those genes of which expression levels change in the brain under ischemic conditions, it is possible to comprehensively identify those genes of which expression levels change under ischemic conditions in tissues other than the brain. More specifically, the hippocampus derived from the above-described mice may be used as a test sample for preparing poly(A)+mRNA. Since cerebral capillaries exist in the hippocampus, blood cells such as erythrocytes, leukocytes and platelets are present there in a mixed state. Therefore, mRNAs from various blood cells may be contained in mixture in poly(A)+mRNA extracted from a hippocampus tissue.

Poly(A)+mRNA may be prepared by obtaining total RNA from test samples by such methods as the guanidine thiocyanate-cesium chloride method [J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd 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)], the guanidine thiocyanate-phenol-chloroform method [P. Chomcznski et al., Anal. Biochem., 162:156 (1987)] or the phenol-SDS method [R.D. Palmiter, Biochemistry, 13:3606 (1974)], loading the resultant total RNA to an oligo-dT cellulose or poly U Sepharose column for specific adsorption of poly(A)+mRNA, and then eluting the poly(A)+mRNA from the column. In particular, when the test sample is a tissue, it is important to perform purification processes accurately because the state of purification of the total RNA or poly(A)+mRNA greatly influences the yield of cDNA, etc.

For example, when poly(A)+mRNA is prepared by the guanidine thiocyanate-cesium chloride method, first, an appropriate amount (e.g., 5 volumes) of guanidine thiocyanate solution is added to a tissue sample. Then, the tissue sample is disrupted using, e.g., a Polytron homogenizer. Sodium N-lauroyl sarcosinate is added to the disrupted tissue to give a desired concentration (e.g., 0.5%) and agitated. The resultant sample is centrifuged (e.g., at 5000 xg for 10 min). The resultant supernatant is layered over a cushion of cesium chloride-EDTA contained in a centrifuge tube and subjected to ultracentrifugation (e.g., at 100,000 xg for 12 hr). The resultant precipitate is rinsed with 70% ethanol and then dissolved in TE buffer to thereby obtain total RNA. The resultant total RNA is applied to an oligo-dT cellulose column to thereby obtain poly(A)+mRNA.

Alternatively, the preparation of poly(A)+mRNA may be performed using commercial kits. Specific examples of kits for preparing total RNA include RNeasy Total RNA Isolation kit (Qiagen) and TRIzol Reagent (Gibco BRL Life Technologies). Specific examples of kits for isolating poly(A)+mRNA from total RNA include Oligotex Direct mRNA kit (Qiagen) and Oligotex mRNA kit (Quiagen).

(2) Synthesis of cDNA with Reverse Transcriptase

Subsequently, cDNA is synthesized using the poly(A)+mRNA obtained in (1) above as a template. The synthesis of cDNA may be carried out according to the method of Gubler et al. [U. Gubler et al., Gene 25:263 (1987)]. Briefly, oligo(dT)₁₂._lg is added to a solution of poly(A)+mRNA, which is heated and then cooled quickly. To this solution, a single-stranded cDNA synthesis buffer, a dNTP solution (containing mixture of dATP, dGTP, dCTP and dTTP), a ribonuclease inhibitor solution, a dithiothreitol solution, etc. are added and mixed. Then, a reverse transcriptase (e.g., Superscript RT; BRL) is added to the mixture, which is then incubated for a specific period to thereby yield single-stranded cDNA. If necessary, double-stranded cDNA may be synthesized further using the single- stranded cDNA as a template. Briefly, a cDNA synthesis buffer, a dNTP solution (containing mixture of dATP, dGTP, dCTP and dTTP), a dithiothreitol solution, etc. are added to a solution of the single-stranded cDNA and mixed. Then, a DNA polymerase (e.g., T4 DNA polymerase) is added to the mixture, which is then incubated for a specific period to thereby yield double-stranded cDNA. Labeled cDNA may be obtained by using a labeled dNTP (e.g., biotin-labeled dNTP) in the synthesis of single- or double-stranded cDNA.

(3) Preparation of Labeled cRNA Fragments

When a DNA chip on which oligonucleotides are immobilized as DNA probes is used in the method of the invention, labeled cRNA is prepared, if necessary, by in vitro transcription using the cDNA obtained in (2) above as a template. The preparation of labeled cRNA by in vitro transcription may be carried out according to the method of Kreig et al. [Kreig, P.A. et al, Methods in Enzymology 155:397-415 (1987)]. The resultant labeled cRNA molecules must be fragmented before use. The fragmentation of these molecules may be performed by heating in the presence of Mg²⁺ (e.g., at 94°C for 3 min) or by treatment with DNase. The in vitro transcription described above may also be performed using a commercial kit. As an example of in vitro transcription kit, MEGAscript™ In Vitro Transcription Kit (Ambion) may be given.

(4) Hybridization on a DNA Chip

Subsequently, the labeled nucleotide sample obtained in (2) or (3) above is added to a DNA chip to carry out a hybridization reaction. Specific examples of DNA chips useful in the method of the invention include oligoDNA microarray (also called "synthetic-type DNA chip") which is prepared by synthesizing oligoDNAs on a substrate directly, and DNA microarray (also called "paste-type DNA chip") which is prepared by immobilizing pre- synthesized DNAs on a substrate. In the present invention, it is preferable to use a synthetic-type DNA chip that can provide high detection sensitivity, accuracy and reproducibility (e.g., oligoDNA microarray GeneChip™ manufactured by Affymetrix) for identifying genes of which expression levels change under ischemic conditions.

In the examination of gene expression, it is important to carry out hybridization under high stringency conditions to inhibit non-specific bonding. The term "high stringency conditions" refers to those conditions under which hybridization only occurs between two nucleotide strands having 90% or more homology to each other. Stringency may be raised or lowered by changing salt concentrations (e.g., concentrations of NaCl, trisodium citrate) and/or the reaction temperature. The lower the salt concentrations are and the higher the temperatures is, the higher the stringency becomes. Depending on the type of DNA chip used and other factors, a specific temperature and specific salt conditions may be high stringency conditions or low stringency conditions. Thus, high stringency conditions and low stringency conditions should be decided for each chip to be used. With respect to GeneChip™ Mu6500 used in the present invention, high stringency conditions refer to reaction temperatures ranging from 43 to 65°C, preferably 45°C, and Na⁺ concentrations ranging from 500 to 1000 mM, preferably 1000 mM.

(5) Detection and Data Analysis

The double-strands formed on the microarray as a result of the hybridization are analyzed with a fluorescence image scanner or the like. The fluorescence intensities may be measured automatically with a system integrating a fluorescent laser microscope, a CCD camera and a computer. Preferably, a scanner is used which is capable of quantitatively discriminating spots having a size of several ten micrometers and having a distance of approx. 10 m between every two spots. Further, it is preferable that the scanner be capable of handling a plurality of labels and scanning over a wide range at a high speed, and that the scanner be equipped with an automatic focusing function which allows the scanner to manage microscopic distortion in the substrate. As a specific example of a scanner equipped with such a function, GMS 418 Array Reader (Genetic MicroSystems) may be given. The software to be used for the analysis of the above data is, preferably, capable of performing complicated analysis of a large number of oligonucleotides with partially overlapped sequences, such as analysis of mutation or polymorphism.

Alternatively, a commercial system may be used in the present invention which is integrating a set of components necessary for gene analysis using a DNA chip. These components include (i) a DNA chip, (ii) a device for automatically washing and staining the DNA chip after hybridization, (iii) a scanner which reads fluorescence emission, and (iv) a work station which processes and analyzes the information read. As a specific example of such a system, the GeneChip™ analysis system created by Affimetrix may be given. This system is provided with GeneChip™ Laboratory Information Management System (LIMS™) and GeneChip™ Expression Data Mining Tool (EDMT™) as bioinformatics tools for efficient utilization of obtained genetic data. These tools make it possible to output obtained data to SQL compliant databases of GATC (Genetic Analysis Technology Consortium) format to thereby link the system to public genetic information databases on the web. By using this analysis system, more efficient and more extensive data analysis can be made.

The nucleotide sequences of those genes which have been found by the present invention to show altered expression levels under ischemic conditions in mouse, and the amino acid sequences encoded by those genes are shown in SEQ ED NO: 1 through SEQ ED NO: 1066. Since both mouse and human belong to mammals, they are highly similar to each other genetically. Thus, genes which are functionally equivalent to the genes having the nucleotide sequences shown in SEQ ED NO: 1 through SEQ ED NO: 1066 or the genes encoding the amino acid sequences shown in SEQ ID NO: 1 through SEQ ED NO: 1066 may exist in human cells. Accordingly, by measuring expression levels of such human genes, it is possible to perform examination of ischemic conditions on human-derived samples. The term "functionally equivalent genes" used herein includes, in addition to the genes consisting of any of the nucleotide sequences shown in SEQ ED NO: 1 through SEQ ED NO: 1066 themselves or the genes encoding any of the amino acid sequences shown in SEQ ED NO: 1 through SEQ ED NO: 1066 themselves, those genes which have homology to the above genes and play roles identical or similar to the roles of the above genes in the living body. Nucleotide sequence information, amino acid sequence information, etc. on those genes in human cells which are functionally equivalent to mouse-derived genes can be obtained from known databases such as GenBank by searching with keywords such as a part of nucleotide sequence of interest, a part of amino acid sequence of interest, or a gene product name.

It is possible to identify ischemia marker genes of which expression levels change specifically under ischemic conditions. This identification can be performed by further examining the expression levels under other diseases of the above-described genes which were found to show altered expression levels under ischemic conditions.

2. DNA Chips for Examining Ischemic Conditions

DNA chips carrying as DNA probes a part or all of the genes identified in Section 1. above (which show altered expression levels under ischemic conditions) immobilized on their substrates can be used as a DNA chip for examining ischemic conditions. In particular, a DNA chip as shown in Fig. 2 on which three groups of genes (i.e., genes showing high expression levels under ischemic conditions; genes showing moderate expression levels under ischemic conditions; and genes showing low expression levels under ischemic conditions) are located separately may be used as a DNA chip that is capable of evaluating the extent of progress of ischemic conditions. There are two types of DNA chips. One is paste-type chips prepared by immobilizing pre-synthesized DNA probes on their substrates; and the other is synthetic-type chips prepared by synthesizing DNA probes on their substrates directly. The term "DNA probes" used herein refers to DNA strands which are immobilized on the substrate of a DNA chip in order to detect those genes having DNA strands with specific nucleotide sequences. Hereinbelow, processes for preparing both types of DNA chips will be described specifically. (1) Method of Preparing Paste-Type DNA Chips

First, as DNA probes, a part or all of the genes identified in Section 1. above which show altered expression levels under ischemic conditions are prepared by PCR or chemical synthesis. DNA probes must be present on the substrate of a DNA chip as single-stranded DNAs so that they can hybridize with target nucleotide strands having sequences complementary to the sequences of the DNA probes when the target strands access to the DNA probes. Thus, in designing DNA probes, it is desirable to select sequences so that formation of secondary structures that would inhibit the hybridization with target nucleotide strands will occur as little as possible. The term "secondary structures" used herein refers to the stem-loop structure, hairpin structure or the like which is formed by hybridization of a part of a probe with another part of the same probe when the probe has been folded back. Whether sequences of interest would form secondary structures or not can be analyzed using a commercial gene analysis software (e.g., DNASIS; Hitachi Software Engineering).

The preparation of DNA probes by PCR may be carried out by conventional methods [see, for example, Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press (1989)] using, as a template, geno ic DNA, total RNA, mRNA or cDNA derived from an organism to be tested. For example, the gene consisting of the nucleotide sequence shown in SEQ ED NO: 1006 can be used as a marker for examining ischemic conditions since its expression increases remarkably under ischemic conditions. A DNA probe to detect this gene may be obtained by PCR using sense primer 5'-atgctcttccgagctgtgct-3' (SEQ ED NO: 1067), anti-sense primer 5'-cagctcagttgaacgccttt-3' (SEQ ED NO: 1068) and, as a template, cDNA prepared from mRNA derived from mouse hippocampus under ischemic conditions. Whether the amplified fragment by PCR is the fragment of interest or not may be determined by subcloning the amplified fragment into an appropriate vector such as pBlueScriptSK(+) (Stratagene) or pCR2.1 (Invitrogen) and then determining the nucleotide sequence thereof. The nucleotide sequence may be determined by conventional methods such as the chemical modification method by Maxam-Gilbert or the dideoxynucleotide chain termination method using Ml 3 phage. Usually, the nucleotide sequence may be determined using an automated DNA sequencer (e.g., 373A DNA sequencer; Perkin-Elmer).

On the other hand, the preparation of DNA probes by chemical synthesis may be carried out according to conventional DNA synthesis methods used in the art, e.g., the phosphoramidite method, or the phosphonate method. For example, when DNA probes are synthesized by the phosphoramidite method, a nucleoside derivative obtained by introducing a trivalent phosphoramidite residue into the hydroxyl group at 3 '-position of the sugar moiety is used as a synthesis unit. Fist, this amidite unit is activated with 1H- tetrazol and reacted with the 5'-hydroxyl of a DNA strand on a solid phase (step 1), to thereby yield a trivalent phosphite ester. Subsequently, the trivalent phosphite ester is led to a pentavalent phosphate triester through oxidation (step 2), capping (step 3) and liydrogenation (step 4). Then, steps 1 to 4 are repeated. Finally, an oligomer block having the desired nucleotide sequence is cleaved from the solid phase and deprotected to thereby yield the DNA strand of interest.

Subsequently, the thus obtained DNA probe is immobilized on the substrate of a DNA chip. Specific examples of substrates useful for this purpose include glass sheets, quartz sheets and silicone wafers. As a size of the substrate, 3.5 mm x 5.5 mm, 18 mm x 18 mm or 22 mm x 75 mm may be used, for example. This size may be varied appropriately depending on, for example, the number and size of spots of DNA probes on the substrate. As to a method for immobilizing DNA, DNA may be electrostatically bound to a solid support that has been surface-treated with a polycation such as polylysine, polyethyleneimine or polyalkylamine, utilizing the electric charge of the DNA; or DNA probes into which a functional group such as amino group, aldehyde group, SH-group or biotin has been introduced may be covalently bound to the surface of a solid support into which a functional group such as amino group, aldehyde group or epoxy group has been introduced.

The spotting of DNA probes on the substrate may be performed using an arrayer which is capable of quantitatively spotting DNA probes in sizes ranging from several ten micrometers to several hundred micrometers and at pre-determined locations. As to the technology of spotting, pin technology utilizing the mechanical contact of pin tips with a solid support; inkjet technology utilizing the principle of inkjet printer; or capillary technology utilizing a capillary device may be enumerated.

(2) Method for Preparing Synthetic-Type DNA Chips

As a method for synthesizing DNA probes on a substrate directly, the method of Fodor et al. may be used in which photolithographic fabrication techniques are combined with solid phase DNA synthesis techniques [Foder, S.P.A. et al, Science 251:767-773 (1991)]. Briefly, a synthetic linker having a protective group removable by a photochemical reaction is bound onto a substrate. Then, the substrate is illuminated by light through a blocking material called mask to thereby remove only those protective groups in specific areas. Subsequently, the substrate is reacted with nucleotides having protected hydroxyl groups. As a result, polymerization occurs only in those areas where protective groups have been removed. Then, the substrate is illuminated by light through another mask, and polymerization of nucleotides is repeated. Thus, coupling reactions with different nucleotide precursors are repeated using various masks. As a result, DNA probes of desired sequences can be synthesized on specific areas on the substrate of a DNA chip. An oligonucleotide N-mer in nucleotide length can be synthesized by Nx4 cycles of reaction. Thus, a DNA probe 25-mer in length can be synthesized by 25 x 4 = 100 cycles of reaction. The nucleotide length of the DNA probes on the DNA chip of the invention for examining ischemic conditions is 10- to 30-mer, preferably 15- to 25-mer.

Since the nucleotide length of DNA probes on DNA chips of this type is usually short, the specificity of hybridization on such chips may be questioned. This problem can be solved as described below. Briefly, in order to detect the expression of a particular gene, perfect match (PM) (i.e., completely complementary) oligonucleotide DNA probes corresponding to a plurality of portions (usually, ten and several portions) of the target gene and an identical number of mismatch (MM) oligonucleotide DNA probes having a mutation at one nucleotide (usually, the central nucleotide or neighboring nucleotide) are located on a substrate (see Fig. 3). Then, hybridization is carried out on the substrate using the MM probes as an indicator of the specificity of hybridization. That is, signal ratio of PM probes to MM probes is calculated, and the pseudo-positive signal is eliminated.

3. Method of the Invention for Examining Ischemic Conditions

Ischemic conditions can be examined by measuring the expression levels in a test sample of the genes which were revealed in the present invention to show altered expression levels under ischemic conditions. Alternatively, ischemic conditions can be examined by determining the expression profile of a gene group comprising a plurality of genes selected from the above-described genes. The expression "determining the expression profile of a gene group" means measuring the expression levels of individual genes constituting the group and arranging the results in tables, graphs, or the like. Specific examples of the ischemic conditions include compressive ischemia, occlusive ischemia and vasospastic ischemia.

By measuring the expression levels in a test sample of the genes identified in Section 1. above which show altered expression levels under ischemic conditions, it is possible to examine whether the test sample is under ischemic conditions or not. Briefly, when the expression levels of the above genes in the test sample are changed to the same extent as the expression levels under ischemic conditions revealed by the present invention are changed, then the test sample can be evaluated as being under ischemic conditions. For example, the expression levels of the genes having the nucleotide sequences shown in SEQ ED NOS: 960-1037 and SEQ ED NOS: 1065-1066 or the genes encoding the amino acid sequences shown in SEQ ED NOS: 960-1037 and SEQ ED NOS: 1065-1066 increase more than 10-fold under ischemic conditions. Thus, when the expression levels of these genes are increased more than 10-fold in a test sample, the test sample can be evaluated as being under ischemic conditions.

Further, it is possible to more accurately examine whether a test sample is under ischemic conditions or not by measuring the expression levels of ischemia marker genes, which are genes included in the genes identified in Section 1. above and show little or no changes in expression levels in diseases other than ischemia. That is, when the changes in the expression levels of the above marker genes in a test sample are the same in extent as the changes in the expression levels of those genes detected in the present invention, the test sample can be evaluated as being under ischemic conditions.

The expression levels of the above genes may be measured by, for example, dot hybridization, slot hybridization, Northern hybridization or quantitative PCR when the number of genes to be measured is small. When the number of genes to be measured is large, their expression levels may be measured with a DNA chip.

Further, it is possible to examine with higher accuracy whether a test sample is under ischemic conditions or not by determining the expression profile of a gene group in the sample comprising a plurality of genes selected from the genes identified in Section 1. above which show altered expression levels under ischemic conditions and comparing the resultant profile with an expression profile of a normal sample which is not under ischemic conditions. The determination of expression profiles of gene groups can be performed more quickly and simply by using DNA chips. Expression profiles may be classified using cluster analysis described later. As to the DNA chip, it is preferable to use synthetic-type DNA chips from the viewpoints of accuracy, sensitivity and reproducibility. It is also possible to perform examination of ischemic conditions using the DNA chip of the invention prepared in Section 2. above. For example, as shown in Fig. 2A, genes are classified into a group of low expression level genes, a group of moderate expression level genes, and a group of high expression level genes and immobilized separately on a DNA chip for hybridization. Low expression level genes mean those genes of which transcription levels increased n-fold (where n is 2 or more but less than 5) within 24 hours when the transcription level at 0 hour is taken as 1. Moderate expression level genes mean those genes of which transcription levels increased n-fold (where n is 5 or more but less than 10). High expression level genes mean those genes of which transcription levels increased more than 10-fold.

In order to prepare a DNA chip carrying immobilized genes thereon which show altered expression levels under ischemic conditions, for example, 300 or more low expression level genes, 100-300 moderate expression level genes and 30-100 high expression level genes are selected from the genes having the nucleotide sequences shown in SEQ ID NO: 1 through SEQ ID NO: 1066 or the genes encoding the amino acid sequences shown in SEQ ID NO: 1 through SEQ ID NO: 1066, and immobilized. When the expression levels of the 30-100 high expression level genes have been changed compared with a gene expression profile obtained form a non-ischemic patient (control), the test sample can be judged under ischemic conditions of early-stage (Fig. 2B). When not only the expression levels of high expression level genes but also those of the 100-300 moderate expression level genes have been changed, the sample can be judged under ischemic conditions of intermediate-stage (Fig. 2C). Further, when the expression levels of the 300 or more low expression level genes have been changed in addition to those of the high expression level genes and the moderate expression level genes, the test sample can be evaluated under ischemic conditions of late-stage (Fig. 2D).

Expression levels may change toward increase or decrease compared to normal levels. However, the expression levels of all of the genes having the nucleotide sequences shown in SEQ ID NO: 1 through SEQ ID NO: 1066 or the genes encoding the amino acid sequences shown in SEQ ID NO: 1 through SEQ ID NO: 1066 change toward increase under ischemic conditions. Thus, in examining ischemic conditions using changes in the expression levels of the above genes as an indicator, a gene should be counted as having shown a change in its expression level only when an increase is observed in the expression level.

With respect to those genes which have been confirmed to show no change in expression levels under ischemic conditions, it is also possible to measure their expression levels and utilize the results for examining ischemic conditions. Briefly, the expression levels of such genes are measured in the test samples which were evaluated as ischemic as a result of the above-described measurement. If no change is observed in their expression levels, the reliability of the above evaluation that the test samples are under ischemic conditions can be enhanced.

4. Computer-Readable Record Medium Containing Gene Expression Data under Ischemic Conditions and Ischemic Condition Identification Program, as well as Ischemic Condition Identification Program

Expression level data of the genes which were revealed by the invention to show altered expression levels under ischemic conditions or expression profile data of a gene group comprising a plurality of genes selected from the above genes may be recorded in an appropriate medium and used as comparison data in the analysis of examination data on ischemic conditions. As a record medium, any type of record media may be used, e.g., magnetic tape, CD-ROM, IC card, or RAM. Specifically, the degree of change in the expression levels of those genes of which expression levels change remarkably under ischemic conditions is examined in a test sample. If the expression levels are equal to the expression levels recorded in a record medium, the test sample (and thus the organism from which the sample is derived) can be evaluated as being under ischemic conditions. Further, more accurate evaluation of ischemic condition can be made by comparing a gene expression profile recorded (which was created from individual expression level data of a group of genes whose expression levels change under ischemic conditions) with an expression profile of corresponding genes in a test sample. That is, if the expression patterns in a test sample of a plurality of particular genes whose expression levels change under ischemic conditions resemble the expression patterns of the corresponding genes recorded in such a medium, the test sample can be evaluated as being under ischemic conditions with higher probability.

A record medium in which a program that directs a computer to execute the procedures described below has been recorded is useful as a record medium containing ischemic condition identification program. The term "ischemic condition identification program" used herein refers to a program that is able to identify the stage of ischemic conditions (i.e., early stage, intermediate stage or late stage) in a test sample when the test sample has been suspected to be under ischemic conditions or evaluated as being under such conditions. This program comprises (a) procedures to input expression level data or expression profile data of a test sample; (b) procedures to record the input data; (c) procedures to check this recorded data with already recorded expression level data or expression profile data under ischemic conditions; (d) procedures to determine whether the test sample is under ischemic conditions or not based on the checking results obtained in (c); and (e) if the test sample has been determined as being under ischemic conditions, procedures to identify the clinical stage of the ischemic conditions of the test sample based on the checking results obtained in (c). By analyzing the gene expression data of a test sample using a computer in which the above-described problem has been installed, ischemic conditions can be identified.

The ischemic condition identification program of the invention comprises (a) means for analyzing expression levels of genes isolated from test cells; and (b) means for predicting whether or not individual test samples are under ischemic conditions or not, using the analysis results obtained by (a) as an indicator. The analysis means (a) is composed of means for detecting individual expression levels of a plurality of genes in test cells or tissues (also called "detection engine"), and means for analyzing the resultant values detected (also called "analysis engine").

(1 ) Detection Engine for Gene Expression

In the present invention, when expression of genes has been detected, the detection data may be digitalized and used as digital information. The digitalization is performed by converting, for example, fluorescence intensities detected on DNA chips into numerical values.

(2) Analysis Engine

Analysis engine is a means for performing analysis processing by multivariate analysis such as cluster analysis, based on the data (i.e., amounts of gene expression) obtained by the detection engine. Cluster analysis, which is a technique used in the field of multivariate analysis, collects and classifies "similar objects" from a large number of objects (i.e., samples) to be observed based on specific calculation criteria (assess criteria). In other words, cluster analysis merely "classifies" a large number of samples observed by putting samples similar to each other into one group.

In order to perform cluster analysis based on the detection data, "distance matrices" that represent similarities between samples are created. As the distance, Euclidean distance, weighted Euclidean distance, standard Euclidean distance, Pearson's product- moment correlation coefficient, or the like is calculated. The concepts of these distances are known, and an appropriate distance may be selected depending on the purpose of cluster analysis. Based on the concept of the above-mentioned distance, distances between clusters or distances between a cluster and objects are calculated, followed by amalgamation of clusters (i.e., two clusters are linked together). Methods of amalgamation are known, e.g., the nearest neighbor method, furthest neighbor method, centroid method, or Ward's method.

By the above-described procedures, clusters which are in the "shortest distance" relation are linked together as "similar" clusters to thereby generate new clusters of a higher level. When clusters at one level have been generated, distances between clusters are calculated again to create distance matrices. Then, by searching for two clusters at the shortest distance, new clusters at a higher level are generated. Thus, a dendrogram is created finally.

Samples within a cluster amalgamated at a specific level of a dendrogram are contained in that cluster because of some similarity. Those samples with such similarity can be said to possess a certain nature in common. By elucidating this nature, it is possible to reveal the characteristic of the cluster itself. Thus, according to these analysis procedures, it is possible to classify genes into a group of high expression genes and a group of low expression genes. For example, if focusing on the degree of ischemic conditions using the progress (stage) of ischemia as an indicator, it is possible to reveal such characteristics that samples belonging to one cluster are under highly ischemic conditions and that samples belonging to the other cluster are under lightly ischemic conditions.

One embodiment of the identification system of the invention is illustrated in a block diagram (Fig. 5).

The identification system shown in Fig. 5 is equipped with CPU 501, ROM 502, RAM 503, Input Unit 504, Sending/Receiving Unit 505, Output Unit 506, Hard Disk Drive (HDD) 507 and CR-ROM Drive (508).

CPU 501 controls the ischemic condition identification system entirely and executes the examination processing described below according to the programs stored in ROM 502, RAM 503 or HDD 507. ROM 502 contains programs, etc. that instruct processing necessary for the operation of the above system. RAM 503 contains those data necessary for executing the examination processing. The Input Unit 504 is composed of a keyboard, mouse, etc. and operated, e.g., for inputting necessary conditions for the execution of the examination processing. The Sending/Receiving Unit 505 executes, based on instructions from CPU 501, the sending/receiving of data to/from External Database 510, etc. through communication circuits. The Output Unit 506 executes display processing of various conditions input from the Input Unit 504, detection data on expressed genes, etc. based on instructions from CPU 501. The Output Unit 506 may include a computer-display unit and a printer. HD 507 contains information of expression patterns of various genes in cells or tissues and, based on instructions from CPU 501, reads out stored programs or data and stores them, e.g., in RAM 503. Based on instructions from CPU 501, CR-ROM Drive 508 reads out programs or data from the identification program stored in CD-ROM 509 and stores them, e.g., in RMA 503.

CPU 501 executes prediction of whether individual test samples are under ischemic conditions or not based on the data received from the Database, while supplying data received from the Input Unit, etc. to the Output Unit 506. The Database contains accumulated information of the amounts of gene expression (including both absolute amounts and relative amounts) obtained as described above.

Fig. 6 is a flow chart showing an example of identification processing using the identification program described above. Expression patterns of genes in test samples are analyzed, followed by identification of whether individual samples are under ischemic conditions or not .

Hereinbelow, the identification processing will be described with reference to Cluster Analysis Device 601 in Fig. 6. Cluster Analysis Device 601 generates clusters to be used in the identification processing. First, gene expression data are input by Means for External Database Searching and Data Input 602. Until data input is completed, input operation of the above data is repeated. By the input of the above data, information obtained from each tissue or cell is stored in Sample Data Storage Means 603, and supplied for cluster analysis or registered in the database.

Subsequently, Data Optimizing Means 604 inputs sample data from Sample Data Storage Means 603 and optimizes the data for cluster analysis. Data optimization is performed using a method most suitable for the sample, e.g., normalization with median values, normalization with z-scores, setting the maximum and the minimum values, or log transformation. Means for Outputting List of Variables 605 displays a list of variables in the sample data to be subjected to cluster analysis.

Subsequently, using the function of Variable Selection Means 606, a user selects variables from the variables displayed by Means for Outputting List of Variables 605.

The selection of variables by Variable Selection Means 606 allows free selection of a single or a plurality of particular variables. Since, usually, there are a large number of candidates for variables, the Means 606 allows the user selection of any variables from them.

Once the user has selected particular variables, this information is input into Means for Generating Sample Data Files for Identification 607 together with sample data. Then, sample data files for identification is generated by this Generating Means 607.

Subsequently, the resultant data files of clusters are sent to Identification Means 608, which evaluates the degree of separation of clusters. The evaluation formula to evaluate the degree of separation of clusters may be defined in various manners. The results of evaluation of the degree of separation made by the Identification Means 608 are passed to Means for Classifying Clusters 609. Then, the Classifying Means 609 inputs the identification results by the Identification Means 608 and decides most appropriate cluster classification referring to the identification conditions set in Means for Setting Identification Conditions 612. If conditions for continuation/termination of cluster classification have been set, the Classifying Means 609 judges the continuation or termination of cluster classification. If conditions for continuation/termination of cluster classification have not been set, the Classifying Means 609 allows the user to judge the continuation or termination of cluster classification. If the Classifying Means 609 has decided to continue cluster classification, it outputs the most appropriate cluster classification obtained from the processing of that time and a signal announcing that cluster classification is continued. This signal will work later as an instruction that processing of cluster classification must be returned to the Means for Outputting List of Variables 605 after the processing by Means for Editing Dendrograms 611.

On the other hand, if the Classifying Means 609 has decided to terminate cluster classification, it specifies the most appropriate cluster at that stage and outputs a signal announcing that cluster classification is terminated. This signal will work later as an instruction to terminate the processing of cluster classification after the processing by Means for Editing Dendrograms 611.

After completion of the processing by the Classifying Means 609, the processing by Means for Generating Dendrograms 610 starts. The Generating Means 610 inputs the cluster classification decided by the Classifying Means 609, and displays a dendrogram based on the above cluster classification and characters of the variables pertaining to the cluster classification. When the Generating Means 610 has generated this cluster classification dendrogram, the user becomes able to grasp the current state of cluster classification visually. While generating the dendrogram, the Generating Means 610 displays the amounts of gene expression which are basis for the generation of the dendrogram so that the user can grasp the amounts visually. Then, Means for Editing Dendrograms 611 allows the user on the screen of a display device to edit (i.e., make additions, changes, or deletions to) the dendrogram generated by the Generating Means 610. The addition, change, or deletion of cluster classification is performed by the user with a processing instruction input device on the screen. For example, certain clusters may be designated, and variables of clusters to be classified further at a lower level may be indicated; or a plurality of clusters may be amalgamated. Alternatively, branches of a certain cluster classification may be deleted. While providing various tools to support the user's editing operation, Means for Editing Dendrograms 611 reads the meaning of the user's editing operation and automatically corrects data files of individual clusters accordingly. Furthermore, Means for Editing Dendrograms 611 preferably presents the judgment of Cluster Classifying Means 609 to continue or terminate the cluster classification and allows the user to input his/her final decision.

If it is decided that repeated processing of cluster classification should be continued, the processing is returned to Means for Outputting List of Variables 605. Then, the above- described processing performed by Means for Outputting List of Variables 605 through Means for Editing Dendrograms 611 is repeated.

From the thus analyzed data, whether the test samples are under ischemic conditions or not can be judged by checking into which cluster (i.e., ischemic cluster or normal cluster) they have been classified.

5. Method of Screening for Ischemic Condition-Improving Drugs or Therapeutics for Ischemic Diseases It is possible to screen for ischemic condition-improving drugs or therapeutics for ischemic diseases using as an indicator the expression levels of the genes which were revealed in the invention to show increased expression levels under ischemic conditions. Briefly, (a) whether or not the expression levels of the above genes return to their expression levels in a normal tissue, or (b) whether or not the expression profile of a gene group comprising a plurality of above genes returns to the normal expression profile in a normal tissue, by the administration of a drug to a test animal or test cell is examined. When the expression levels of the above genes have returned to their expression levels in a normal tissue as a result of the administration of the drug, the drug is evaluated as a candidate substance for an ischemic condition-improving drug or therapeutic for ischemic diseases.

BRIEF DESCRIPTION OF DRAWINGS

Fig. 1 shows procedures to identify genes of which expression levels change under ischemic conditions using a DNA chip.

Fig. 2 shows one embodiment of the DNA chip of the invention for examining ischemic conditions, and predicted results when test samples from patients have been hybridized on the DNA chip.

Fig. 3 shows one embodiment of synthetic-type DNA chip and procedures to detect genes in a test sample using the DNA chip.

Fig. 4 shows procedures to measure the expression levels of genes using GeneChip™.

Fig. 5 is a block diagram showing an ischemic condition identification system. Fig. 6 is a flow chart showing an example of processing by an ischemic condition identification program. Legend

501: CPU; 502: ROM; 503: RAM, 504: Input Unit; 505: Sending/Receiving Unit; 506: Output Unit; 507: HDD, 508: CD-ROM Drive; 509: CD-ROM; 510: Database; 601: Cluster Analysis Device; 602: Means for External Database Searching and Data Input; 603: Sample Data Storage Means; 604: Data Optimizing Means; 605: Means for Outputting List of Variables; 606: Variable Selection Means; 607: Means for Generating Sample Data File for Evaluation; 608: Evaluation Means; 609: Means for Classifying Clusters; 610: Means for Generating Dendrograms; 611: Means for Editing Dendrograms; 612: Means for Setting Evaluation Conditions.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinbelow, the present invention will be described more specifically with reference to the following Example, which should not be construed as limiting the scope of the invention.

Example 1. Identification of Genes Expressed under Ischemic Conditions using a DNA Chip

Genes expressed under ischemic conditions were identified by the procedures as shown in Fig. 4 using GeneChip System™ (Affymetrix). Briefly, 8-10 week-old bcl BLACK mice were anesthetized with inflane. Then, the carotid artery on both sides was exposed and ligated for 20 min to block the blood flow to thereby generate ischemic conditions in mice. Then, the blood flow was restored, and mice were slaughtered at 0, 2, 6, 12 and 24 hours after the blood flow restoration. The hippocampus on both sides was removed and immediately stored frozen. Subsequently, approximately 2 Id g of poly(A)+mRNA was extracted from 1 g of a frozen sample. Then, cDNA was synthesized with a reverse transcriptase. The resultant cDNA was transcribed in vitro to thereby obtain biotin-labeled cRNA, which was treated with heat in the presence of Mg²⁺ for fragmentation into about 50-mer fragments. Internal reference was labeled and added to the sample. Then, the sample was poured into GeneChip™ Mu6500 (Affymetrix). After hybridization in an oven, the chip was washed in Fluidic station. Then, information of the chip was read by a GeneArray scanner. The data obtained was processed and analyzed using a bioinformatics system. The results are shown in Tables 1 through 3. Table 1 shows genes of which transcription levels increased n-fold where n is 2 or more but less than 5 within 24 hours when the transcription level at 0 hour is taken as 1. Table 2 shows genes of which transcription levels increased n-fold where n is 5 or more but less than 10. Table 3 shows genes of which transcription levels increased more than 10-fold. The accession numbers refers to GenBank accession numbers.

Table 1.

Table 2.

Genes of Which Transcription Levels Increased n-Fold (5≤n< lO)

Table 3.

All the publications, patents and patent applications cited in the present specification are incorporated herein by reference in their entireties.

INDUSTRIAL APPLICABILITY

According to the present invention, ischemic conditions are examined by analyzing gene expression in a test tissue. Application of such analysis to prevention and treatment of ischemia is expected. There is also provided a novel method of screening for prophylactics and therapeutics for ischemia using, as an indicator, the expression levels of genes which are expressed specifically under ischemic conditions.

SEQUENCE LISTING FREE TEXT

SEQ ID NO: 1067: synthetic DNA SEQ ID NO: 1068: synthetic DNA

Claims

1. A method for examining ischemic conditions, comprising measuring the expression levels of particular genes in a test sample or determining the expression profile of a gene group in the sample comprising a plurality of genes selected from said particular genes.

2. The method according to claim 1, wherein said particular genes are:

(a) genes having any of the nucleotide sequences shown in SEQ ID NO: 1 through SEQ ID NO: 1066 or genes encoding any of the amino acid sequences shown in SEQ ID NO: 1 through SEQ ID NO: 1066; or

(b) genes functionally equal to the genes having any of said nucleotide sequences or genes functionally equal to the genes encoding any of said amino acid sequences.

3. The method according to claim 1 or 2, wherein the measurement of the expression levels or the determination of the expression profile is carried out with a DNA chip.

4. The method according to claim 3, wherein the DNA chip is a synthetic-type DNA chip.

5. 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, occlusive ischemia and vasospastic ischemia.

6. A DNA chip for examining ischemic conditions, carrying a part or all of the following genes (a) or (b) immobilized on its surface:

(a) genes having any of the nucleotide sequences shown in SEQ ID NO: 1 through SEQ ID NO: 1066 or genes encoding any of the amino acid sequences shown in SEQ ID NO: 1 through SEQ ID NO: 1066; or (b) genes functionally equal to the genes having any of said nucleotide sequences or genes functionally equal to the genes encoding any of said amino acid sequences.

7. The DNA chip according to claim 6, wherein the ischemic condition is at least one selected from the group consisting of compressive ischemia, occlusive ischemia and vasospastic ischemia.

8. A method of screening for ischermc condition-improving drugs or therapeutics for ischemic diseases, comprising selecting candidate drugs using as an indicator whether or not:

(b) the expression profile of a gene group comprising a plurality of said particular genes returns to a normal expression profile; by the administration of a drug to a test animal or test cell, wherein the returning to the normal expression levels or normal expression profile indicates that said drug is a candidate drug.

9. The method according to claim 8, wherein said particular genes of which expression levels change under ischemic conditions are:

10. The method according to claim 8 or 9, wherein the ischemic condition is at least one selected from the group consisting of compressive ischemia, occlusive ischemia and vasospastic ischemia.

11. A computer-readable record medium in which the following data (a) or (b) have been recorded:

(a) expression level data of genes of which expression levels change under ischemic conditions; or

(b) expression profile data of a gene group comprising a plurality of genes selected from said genes.

12. The record medium according to claim 11, wherein said genes of which expression levels change under ischemic conditions are:

(b) genes functionally equal to the genes having said nucleotide sequences or genes functionally equal to the genes encoding said amino acid sequences.

13. The record medium according to claim 11 or 12, wherein the ischemic condition is at least one selected from the group consisting of compressive ischemia, occlusive ischemia and vasospastic ischemia.

14. A computer-readable record medium in which a program that directs a computer to execute the following procedures has been recorded:

(b) procedures to record the input data;

(c) procedures to check the recorded data with already recorded expression level data or expression profile data of said genes under ischemic conditions;

(e) if the test sample has been determined as being under ischemic conditions, procedures to identify the clinical stage of the ischermc conditions of the test sample based on the checking results obtained in (c).

15. The record medium according to claim 14, wherein said genes are:

16. The record medium according to claim 14 or 15, wherein the ischemic condition is at least one selected from the group consisting of compressive ischemia, occlusive ischemia and vasospastic ischemia.