WO2003070951A1

WO2003070951A1 - Arteriosclerosis-specific gene and utilization thereof

Info

Publication number: WO2003070951A1
Application number: PCT/JP2003/001926
Authority: WO
Inventors: Osamu Yasuda; Keisuke Fukuo; Toshio Ogiwara
Original assignee: Japan Science And Technology Agency
Priority date: 2002-02-21
Filing date: 2003-02-21
Publication date: 2003-08-28
Also published as: JPWO2003070951A1

Abstract

It is intended to provide a gene which is expressed specifically in arteriosclerosis lesion, a polypeptide and utilization thereof. From arteriosclerosis lesion cells of an apolipoprotein E-lacking mouse (ApoE-knockout mouse), a novel gene expressed specifically in these cells is cloned. Further, a gene homologous to this gene is obtained from a normal tissue. By measuring the expression of the above gene in smooth muscle cells and the formation of a protein encoded by this gene, arteriosclerosis can be diagnosed and a preventive or a remedy for arteriosclerosis can be screened. Moreover, arteriosclerosis can be prevented or treated by inhibiting the expression of the gene in arteriosclerosis smooth muscle cells. By transferring the gene expressed in arteriosclerosis into smooth muscle cells, apoptosis can be induced in vascular cells and thus the onset of arteriosclerosis can be prevented.

Description

Description Arteriosclerosis-specific gene and its utilization

The present invention relates to genes, polypeptides, and uses specifically expressed in atherosclerotic lesions. Background art

Smooth muscle cells are important components involved in vascular wall morphogenesis and pharmacological responses. Normally, smooth muscle cells inside the adult aortic wall do not divide and are thought to be specialized for muscle cell contraction (contractile type). However, in some cases, vascular smooth muscle cells may exhibit some synthetic capacity, extracellular matrix production, and cell division. It is known that smooth muscle cells in atherosclerotic lesions proliferate and migrate and produce extracellular matrix (proliferative type), which is thought to be due to the transition of smooth muscle cells to an abnormal state. Such conversion is important in studying the etiology of atherosclerosis (Nature 362, 801-809, 1990; Proc. Natl. Acad. Sci. USA, 70, 1753-1756, 1973; Circ. Res. 58, 427-444, 1986).

However, the mechanism by which smooth muscle cells transition from a contractile form to a proliferative form is still unclear. Numerous studies have already shown that smooth muscle cells can have the ability to proliferate, and when smooth muscle cells from humans, primates, egrets, or rat arteries are dispersed in culture, they naturally become It is already known to convert to a phenotype capable of synthesizing (Physiol. Rev. 59, 1-61, 1979; Lab. Invest. 33, 16-27, 1975; Cel 1 Tiss. Res. 177, 503). -522, 1977). A recent study showed that isolation of smooth muscle cells It is also known that differentiated (contracting) cells or undifferentiated (proliferating) cells continue to partially maintain their gene expression patterns even after repeated doublings in culture. (Am. J. Pathol. 144, 1068-1081, 1994; Arterioscler. Thromb. 13, 1449-1455, 1993). This indicates that cells with these two phenotypes can stably coexist in normal blood vessel walls, and that cells with a synthetic phenotype proliferate in response to stimuli during the disease process. .

Comparing cells from atherosclerotic and normal arteries, it is known that there is a difference in proliferation rate. For example, when cells obtained from the atherosclerotic artery of the egret or the artery of a rat thickened by balloon scraping are compared with cells derived from a control normal artery, active proliferative activity is observed. (Atherosclerosis 36, 241-248, 1980; Med. Biol. 62, 255-259, 1984; Arch. Pathol. Lab. Med. 112, 987-996, 1988; Artery 14, 266-282, 1987). On the other hand, human (Am J Pathol 78, 175-190, 1975) and Egret (Med Biol 60, 221-225, 1982) were used to analyze cells from arteries with atherosclerotic lesions and cells from normal arteries. It is also known that there is no difference when obtained and compared. Furthermore, examination of atherosclerotic plaque-derived cells obtained from humans has already shown that cell death (apoptosis) is more likely to occur than control-derived cells derived from blood vessels (Circ Res 81, 59, 599). , 1997). No studies have been performed to compare smooth muscle cells from an advanced atherosclerotic lesion and a normal arterial segment in an animal individual. In addition, no studies have been performed comparing black (P) -derived and non-plaque (NP) -derived smooth muscle cells in responding to factors that promote and suppress cell proliferation. Furthermore, the characteristics of smooth muscle cells in advanced atherosclerotic arterial plaques have also been shown to date. No research was done.

In the development of arteriosclerosis, the proliferation of vascular smooth muscle cells plays a central role. However, hitherto, the genes responsible for the activation of these smooth muscle cells have not been clarified.

In recent years, genomic research has focused on the genes responsible for atherosclerosis. In February 1995, the United States DawinMolecular, in partnership with France's RPR Gencell Division, Genethon, France, worked to identify the etiological genes of atherosclerosis by pedigree analysis and positional cloning. It is reported that the search has begun. It was reported that Millenium Pharmaceuticals of the United States and Eli Lily of the United States began screening therapeutic agents based on the causative gene for atherosclerosis in October 1995. .

Recently, it has been reported that the type VIII collagen gene is expressed by vascular smooth muscle cells in response to vascular injury (Circulation Research, 80, 532-541, 1997).

The present inventors proceeded with research to elucidate the expression of genes responsible for the activation of smooth muscle cells, and found that one of these genes is the type 1 VIII collagen gene. This was published (J. Vase. Res. 37, 158-169, 2000).

It is an object of the present invention to provide a gene and polypeptide specifically expressed in atherosclerotic lesions, and a gene and polypeptide homologous to the gene and polypeptide expressed in normal vascular smooth muscle cells. And to provide their use. Disclosure of the invention

'' The present inventors have conducted intensive research to solve the above-mentioned problems, and have found that A gene specifically expressed in atherosclerotic lesion cells of a mouse deficient in Epo (Apo E) (apoE knockout mouse) is cloned, and furthermore, in normal vascular smooth muscle cells. The present invention has been completed by cloning a gene homologous to the expressed gene.

That is, the present inventors prepared cultured smooth muscle cells from normal and atherosclerotic lesions, respectively, and conducted a comparative study. As an experimental material, the thoracic aorta was excised from an ApoE knockout mouse, which is a model of spontaneous onset of arteriosclerosis. Cultured smooth muscle cells were prepared from atherosclerotic plaques and normal parts by graft culture. Proliferation rate was measured by [ ³ H] -Thymidine incorporation, and differential expression was used to compare gene expression. As a result, the cultured smooth muscle cells of normal blood vessels and atherosclerotic foci did not show morphological differences. Although the growth rate was different between the cultured cell lines, no significant difference was observed between normal blood vessels and atherosclerotic lesions. There were no significant differences in responsiveness to growth promoting or inhibitory factors.

On the other hand, as a result of comparing gene expression by Differential Display method, differences in expression of several genes were observed. As a result of examining the gene sequence, one of them was type VIII collagen, whose expression was enhanced in atherosclerotic smooth muscle cells. Enhanced expression of collagen VIII was also confirmed in inViVo.

As a result of identification of genes whose expression is enhanced, it was confirmed that unknown genes existed among genes whose expression was enhanced. These new genes were cloned and their structures were determined, one for “F227” (SEQ ID NO: 1 in the sequence listing) and one for “H2A” (SEQ ID NO: SEQ ID NO: 3). Further, from a normal tissue, a gene homologous to the above-mentioned gene expressed in the tissue “A The structures were determined by cloning “K 0 287 3 7” (SEQ ID NO: 5 in the sequence listing) and “NM—003 2 3 7 4” (SEQ ID NO: 7 in the sequence listing).

The arteriosclerosis is measured by detecting the expression of the gene obtained and identified in the present invention and the production of a protein encoded by the gene as a gene marker for diagnosis of arteriosclerosis and a peptide marker. Diagnosis of the disease can be performed, and further, by measuring the expression of the gene and the production of protein, screening for a preventive or therapeutic drug for arteriosclerosis can be performed. Furthermore, by preventing the expression of the gene in arterial smooth muscle cells or by inducing apoptosis by introducing the gene, it is possible to prevent or treat arteriosclerosis.

That is, the present invention relates to a DNA (claim 1) or a DNA comprising a base sequence shown in SEQ ID NO: 1 of the sequence listing or a sequence complementary thereto or a sequence containing a part or all of these sequences. A DNA that hybridizes with the DNA according to claim 1 under stringent conditions and encodes a polypeptide expressed in arteriosclerosis, characterized by DNΑ (claim 2); DNA encoding the polypeptide of a) or (b).

(a) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2 in the sequence listing

(b) a polypeptide comprising an amino acid sequence shown in SEQ ID NO: 2 in which one or several amino acids are deleted, substituted or added, and expressed in arteriosclerosis (claim 3) or a DNA comprising the base sequence shown in SEQ ID NO: 3 in the sequence listing or a sequence complementary thereto, or a sequence containing a part or all of these sequences (Claim 4). A DNA (claim 5), which hybridizes with the DNA according to 4 under stringent conditions and encodes a protein expressed in arteriosclerosis, or the following (a) or (b): A) a DNA encoding the polypeptide; (A) shown in SEQ ID NO: 4 in the sequence listing (B) an amino acid sequence represented by SEQ ID NO: 4 in the sequence listing, wherein one or several amino acids are deleted, substituted or added, and atherosclerosis (Claim 6).

In addition, the present invention relates to a nucleotide sequence of a gene encoding a polypeptide that induces apoptosis in vascular cells, or a complementary sequence thereof, or a part of these sequences, as shown in SEQ ID NO: 5 in the sequence listing. It hybridizes with a DNA characterized by comprising a sequence containing the whole (claim 7) or the DNA according to claim 7 under stringent conditions, is expressed in normal smooth muscle cells, and is used in arteriosclerosis. A gene which has homology to the expressed polypeptide of SEQ ID NO: 2 and encodes a polypeptide which induces apoptosis in vascular cells (Claim 8); A gene encoding the polypeptide of (c) or (d) below. (C) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 6 in the Sequence Listing; (d) one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 6 in the Sequence Listing Which is expressed in normal smooth muscle cells and has homology to the polypeptide of SEQ ID NO: 2 in the sequence listing expressed in arteriosclerosis, and is also apoptotic in vascular cells. , A nucleotide sequence of a gene encoding a polypeptide that induces apoptosis in vascular cells as shown in SEQ ID NO: 7 in the sequence listing, or its complementary sequence, or A DNA comprising a sequence containing a part or the whole of the DNA sequence of claim 10 or a DNA which is hybridized under stringent conditions with the DNA of claim 10; A gene which has homology to the polypeptide of SEQ ID NO: 2 in the sequence listing expressed in the disease and further encodes a polypeptide that induces apoptosis in vascular cells (claim 11). ) Or A gene encoding the polypeptide of (c) or (cl). (C) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 8 in the sequence listing; (d) one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 8 in the sequence listing Which is expressed in normal smooth muscle cells and has homology to the polypeptide of SEQ ID NO: 2 which is expressed in arteriosclerosis. It is composed of a polypeptide that induces cis (Claim 12).

Further, the present invention provides a method for hybridizing with the nucleotide sequence according to claim 1, 4, 7, or 10 under stringent conditions, and in an arterial smooth muscle cell according to claim 1, 4, 7, or 10. An antisense oligonucleotide which suppresses the expression of a gene consisting of the nucleotide sequence of claim 13 (claim 13); and a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 in the sequence listing (claim 14). ) Or the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing, wherein one or several amino acids are deleted, substituted or added, and the amino acid sequence is expressed in arteriosclerosis. A polypeptide having a similar structure (Claim 15); a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 4 in the Sequence Listing (Claim 16); and a polypeptide having SEQ ID NO: 4 in the Sequence Listing. A polypeptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown, and having a similar structure to a protein expressed in arteriosclerosis (claim Item 17), a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 6 in the sequence listing, which is characterized by inducing apoptosis in vascular cells (Claim 18); In the amino acid sequence shown in 6, the amino acid sequence has one or several amino acids deleted, substituted or added, and is expressed in normal smooth muscle cells and expressed in arteriosclerosis. With the polypeptide of SEQ ID NO: 2 A polypeptide having homology and having a structure similar to that of a polypeptide that induces apoptosis in vascular cells (Claim 19); and an amino acid sequence represented by SEQ ID NO: 8 in the Sequence Listing. A polypeptide characterized by inducing one cis or an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 8 in the sequence listing A polypeptide that is expressed in normal smooth muscle cells and has homology to the polypeptide of SEQ ID NO: 2 in the sequence listing expressed in arteriosclerosis, and further induces apoptosis in vascular cells A polypeptide having a structure similar to that of claim 21 or a polypeptide according to any one of claims 14 to 21, and specifically bound to the polypeptide. The antibody (Claim 22), wherein the antibody is a monoclonal antibody, or the antibody (Claim 23), wherein the antibody is a monoclonal antibody. The antibody according to claim 22 (claim 24).

Further, the present invention provides a probe for diagnosing arteriosclerosis having a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence according to claims 1, 4, 7, or 10 (claim 25). A nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence according to claim 1, 4, 7, or 10. All or all of the antisense strand of the nucleotide sequence according to claim 1, 4, 7, or 10. Arteriosclerosis characterized by immobilizing at least one of the probe for diagnosing arteriosclerosis according to claim 25 (claim 26) comprising a part thereof and the DNA according to claim 25 or 26. A microarray or DNA chip for sclerosis diagnosis (claim 27), and an antibody according to any one of claims 22 to 24 and / or a diagnostic probe according to claim 25 or 26. Atherosclerosis diagnostic agent (claim 28) and claims 22.The antibody and Z according to any of claims 24 to 24 or the diagnostic probe according to claim 25 or 26. An arteriosclerosis diagnostic kit characterized by containing (claim 29).

Further, the present invention provides a method for diagnosing arteriosclerosis, which comprises obtaining a sample from a test tissue and measuring the expression of the gene according to claim 1, 4, 7, or 10 in the sample. A method for diagnosing arteriosclerosis, wherein the measurement of the expression of the gene according to claim 30 or claim 30 includes the use of quantitative PCR (claim 31), or a PCR primer. The primers are SEQ ID No. 9 (Forward) in the Sequence Listing and SEQ ID No. 10 (Reverse) in the Sequence Listing, and Z or SEQ ID No. 11 (Forward) in the Sequence Listing and the Sequence Listing. The method for diagnosing arteriosclerosis according to claim 31 (claim 32), and the gene according to any of claims 30 to 32, wherein a primer of the number 12 (Reverse) is used. Is measured using the microarray method, DNA chip method, RT-PCR method, or Northern plotting method. A method for diagnosing arteriosclerosis characterized by the following features: A method for diagnosing arteriosclerosis, characterized by measuring at least one of the polypeptides described in (8) and (8), and using the antibody according to any one of (22) to (24). The method for diagnosing arteriosclerosis according to claim 34, wherein the test substance is administered to an animal model of arteriosclerosis, and SEQ ID NOs: 1, 4, 7 and Expression of one or more genes having the nucleotide sequence described in 10 and one or more polypeptides of the polypeptides described in Z or SEQ ID NOs: 2, 4, 6, and 8 in the sequence listing Atherosclerosis prevention characterized by measuring the production of peptide or its partial polypeptide Or a method for screening a therapeutic agent (Claim 36), or wherein the arteriosclerosis-onset model animal is an ApoE-deficient mouse; SCREENIN The antisense oligonucleotide according to claim 13 is introduced into arterial smooth muscle cells, and the nucleotide sequence according to claim 1, 4, 7, or 10 in arterial smooth muscle cells. A method for suppressing the expression of an atherosclerosis-generating gene characterized by suppressing the expression of a gene consisting of A method for inhibiting arteriosclerosis formation of vascular smooth muscle cells, wherein the method induces apoptosis in vascular cells by introducing the cells into smooth muscle cells, and the vascular smoothing method according to claim 39. A method for gene therapy of arteriosclerosis-onset cells (claim 40), characterized in that smooth muscle cells that have developed arteriosclerosis are treated using a method for inhibiting arteriosclerosis formation of muscle cells. BRIEF DESCRIPTION OF THE FIGURES

Fig. 1 shows X-ray photographs of plaque (P) and non-plaque (NP) smooth muscle cells cultured from three ApoE-deficient mice (# 1-3) and compared for gene expression. FIG.

FIG. 2 is a diagram showing the results of a BLAST homology search performed on the obtained clone F277.

FIG. 3 is a diagram showing the results of a BLAST homology search performed on the obtained clone H2A.

FIG. 4 is a view showing the results obtained by comparing the amino acid sequences encoded by the genes, H 2 A, AK 027 873, and NM-0 32 37 4 obtained in the examples of the present invention. .

FIG. 5 is a photograph taken by a fluorescence microscope of a nucleus stained with a fluorescent dye for morphological analysis of cells that have undergone apoptosis due to the introduction of the gene expressed in arteriosclerosis according to the present invention. . BEST MODE FOR CARRYING OUT THE INVENTION

(Cloning and identification of genes)

Stroke and heart disease, which are the second most common causes of death in Japan, are caused by arteriosclerosis. Therefore, preventing the development and progression of arteriosclerosis is one of the most important clinical issues. In order to elucidate the mechanism of the onset and progression of atherosclerosis, it is important to obtain and clone genes that are expressed in atherosclerotic lesions. In this study, the present inventors isolated and cultured smooth muscle cells from atherosclerotic foci and normal blood vessels from Ap0E knockout mice, which are spontaneous models of arteriosclerosis, and developed a differential display (Differential Display). To compare gene expression. As a result, three types of genes whose expression was enhanced in atherosclerotic lesions were obtained, one of which was type VIII collagen. The other two genes were new genes, so their entire sequences were revealed. Separately, a gene homologous to the above gene was obtained from human and mouse heart gene libraries, and the entire sequence was determined.

One of the two novel genes expressed in the atherosclerotic lesions was completely unregistered in the database. This gene is expressed in many tissues other than the arteries, and was presumed to be involved in basic disease states such as tissue maintenance and regeneration. The other is thought to be the product of alternative splicing from a registered gene of unknown function. The two genes were expressed in atherosclerotic lesions and confirmed to be involved in the pathology.

(Gene of the present invention, polypeptide encoded by the gene and antibody thereof)

In the present invention, the novel genes “F227”, “H2A”, and “AK002287” cloned from the atherosclerotic lesion of the ApoE knockout mouse And the structures of “NM-0 03 2 3 7 4” are shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, and SEQ ID NO: 7 in the sequence listing, respectively. The polypeptides (proteins) encoded by the gene are shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, and SEQ ID NO: 8 in the sequence listing. “AK 0 2 8 7 3” and “NM—0 0 3 2 3 7 4” can be searched on the NCBI database overnight. The present invention provides a nucleotide sequence (gene) shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, and SEQ ID NO: 7 or a sequence complementary thereto, or a sequence containing a part or all of these sequences, Furthermore, it contains a DNA sequence that hybridizes with the nucleotide sequence under stringent conditions and encodes a polypeptide expressed in arteriosclerosis.

A DNA encoding the following polypeptide (a) or (b);

(a) a polypeptide consisting of the amino acid sequences shown in SEQ ID NO: 2 and SEQ ID NO: 4 in the sequence listing,

(b) In the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4 in the sequence listing, one or several amino acids are composed of an amino acid sequence in which deletion, substitution, or addition is performed, and are expressed in arteriosclerosis. A polypeptide encoding a polypeptide of the following (c) or (d):

(c) a polypeptide consisting of the amino acid sequences shown in SEQ ID NO: 6 and SEQ ID NO: 8 in the sequence listing,

(d) in the amino acid sequence shown in SEQ ID NO: 6 and SEQ ID NO: 8 in the sequence listing, consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted or added, and expressed in normal smooth muscle cells; A polypeptide that has homology to a polypeptide expressed in arteriosclerosis and induces apoptosis in vascular cells;

Is included. Further, the present invention relates to an antiserum that hybridizes under stringent conditions with the gene having the nucleotide sequence of SEQ ID NO: 1, 3, 5, or 7 in the sequence listing, and suppresses the expression of the gene in arterial smooth muscle cells. It contains sense oligonucleotides.

In the present invention, various DNA sequence mutations can be performed by well-known genetic engineering gene mutation means.

In the above-mentioned nucleotide sequence of the present invention, the conditions for “hybridizing under nucleotide stringent conditions” include, for example, eight hybridizations at 42 ° C., 1 XSSC, 0.1% Washing at 42 ° C with a buffer containing 303 of the following can be mentioned: hybridization at 65 ° C, and buffer containing 0.1 XSSC, 0.1% SDS. The washing treatment at 65 ° C. can be more preferably mentioned. The factors affecting the stringency of the hybridization include various factors other than the above-mentioned temperature conditions, and those skilled in the art can combine the various factors to obtain the hybridization described above. It is possible to realize the same stringency as the stringency of the above. Further, the present invention provides a polypeptide comprising an amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8 in the sequence listing; It is composed of an amino acid sequence in which several amino acids are deleted, substituted or added, and includes a polypeptide having a similar structure to a protein expressed in arteriosclerosis. The polypeptide of the present invention can be obtained by introducing the gene of the present invention into an appropriate expression vector-host cell expression system and expressing it. '

Furthermore, the present invention includes antibodies induced by the polypeptide of the present invention and specifically binding to the polypeptide. The antibody may be a monoclonal antibody And polyclonal antibodies. The antibody can be prepared by a conventional method using the polypeptide of the present invention as an antigen. The antibody of the present invention can be used for detecting the presence or absence of the expression of the present invention gene in smooth muscle cells by antigen-antibody reaction with the polypeptide of the present invention, and can be used for diagnosis of arteriosclerosis. .

(Use of the gene of the present invention and the polypeptide encoded by the gene)

In the present invention, the novel genes "F227" (SEQ ID NO: 1) and "H2A" (SEQ ID NO: 3) cloned from atherosclerotic lesions, and cloned from normal smooth muscle cells “AK0022873” and “NM-03032374” thus obtained can be used as a diagnostic probe for diagnosis of arteriosclerosis by using an antisense strand of the base sequence or the like. it can. The diagnostic probe or the antibody specifically binding to the polypeptide of the present invention can be used as a diagnostic kit for arteriosclerosis.

Further, at least one DNA of the present invention can be immobilized on a device and used as a microarray for diagnosing arteriosclerosis. Genes expressed in atherosclerotic lesions such as collagen type VIII can be fixed to the microarray together. Microarrays can be prepared by standard methods (Science 270, 467-470, 1995, Cell Engineering Separate Volume Genome Science Series II. “DNA microarrays and the latest PCR method” Shujunsha, Ltd. (2000) March 16)).

The method for diagnosing arteriosclerosis according to the present invention comprises measuring the expression of the gene or protein (polypeptide) of the present invention in a smooth muscle cell as a test tissue.

For gene detection, a commonly used microarray method, DNA chip method, Northern blotting method, or the like can be conveniently used. Also, ATAC—A known method for detecting genes such as PCR (adaptor-tagged competitive PCR), Taq Man PCR, EST (expressed sequence tags), and SAGE (serial analysis of gene expression) can be used as appropriate. it can. For the detection of polypeptides, immunoassays using the antibodies of the present invention, for example, immunoassays such as radioimmunoassay (RIA) and enzyme immunoassay (ELISA), and fluorescence immunoassays (FIA) Publicly known immunological measurement method can be used.

Diagnosis of arteriosclerosis in the present invention can also be performed by measuring the blood concentration of a gene product (the polypeptide of the present invention) based on the expression of the gene obtained in the present invention. These diagnostic methods can be applied to quantitative diagnosis of arteriosclerosis.

The method for measuring the expression of the gene or protein (polypeptide) of the present invention in smooth muscle cells, which is used in the method for diagnosing arteriosclerosis in the present invention, can be used for screening for a preventive or therapeutic drug for arteriosclerosis. . To screen for a preventive or therapeutic drug for arteriosclerosis, a test substance is administered to a model animal with atherosclerosis, and the expression of the gene of the present invention and the production of Z or the polypeptide of the present invention are measured. It is done by doing. In the present invention, quantitative PCR can be used for diagnosis of gene expression in diagnosis of arteriosclerosis. When performing the PCR, as primers, for the “H 2 A” gene, SEQ ID NO: 9 (Forward), SEQ ID NO: 10 (Reverse), and “AK 0 0 287 3” in the sequence listing On the other hand, SEQ ID NO: 11 (Forward) and SEQ ID NO: 12 (Reverse) in the sequence listing can be used. As an arteriosclerosis-onset model animal used for screening for a preventive or therapeutic agent for arteriosclerosis, an ApoE-deficient mouse, which is a spontaneous onset model of arteriosclerosis, can be advantageously used. ApoE-deficient (apoE—) mice spontaneously develop atherosclerotic lesions in the arteries (aorta). It develops naturally and these lesions exhibit characteristics similar to human atherosclerotic lesions (Arterioscler Thromb 14, 141-147, 1994).

In the present invention, an antisense oligonucleotide is prepared using the gene of the present invention, which comprises the nucleotide sequence of SEQ ID NO: 1, 3, 5, or 7 in the sequence listing, and the antisense oligonucleotide is subjected to arterial smoothing. It can be introduced into muscle cells to suppress the expression of the atherosclerosis-causing gene, thereby preventing or treating arteriosclerosis.

In order to suppress the expression of the atherosclerosis onset gene, an antisense oligonucleotide is prepared by hybridizing the gene consisting of the nucleotide sequence of SEQ ID NO: 1, 3, 5 or 7 under stringent conditions. And a base sequence having an inhibitory action on the expression of the gene is prepared. The size of the antisense oligonucleotide is not particularly limited as long as it can suppress the expression of the gene.However, in consideration of administration to the living body and stability, etc., 12 to 40 base pairs, particularly 1 to 40 base pairs are considered. Those having about 5 to 25 base pairs are preferred.

For the introduction of the antisense oligonucleotide into arterial smooth muscle cells, a method usually used in this field can be used. For example, antisense oligonucleotides can be administered directly to arterial smooth muscle cells. Further, if necessary, it can be administered together with a pharmaceutically acceptable reagent for introduction into a cell, for example, a lipofectin reagent, a lipofectamine reagent, a DOTAP reagent, a Tfx reagent, a ribosome, a polymer carrier and the like. In this case, the antisense oligonucleotide may be used alone or as a pharmaceutically acceptable carrier, binder, stabilizer, excipient, diluent, PH buffer, solubilizer, solubilizer. And the like can be added.

To administer the antisense oligonucleotide to cells, it can be administered in the form of an injection. In this case, the antisense oligonucleotide It can be prepared by dissolving nucleotide in water, physiological saline, glucose solution, or the like, and administering a buffer, preservative, stabilizer or the like as necessary.

In addition, a plasmid vector virus designed to express an antisense oligonucleotide may be used as a vector for gene therapy in order to promote its incorporation into cells and enhance its directivity to target cells. it can. Examples of the vector include virus vectors such as herpes virus (HSV) vector, adenovirus vector, human immunodeficiency virus (HIV), retrovirus vector and the like.

In addition, the gene represented by SEQ ID NO: 3, 5, or 7 in the sequence listing of the present invention and a mutant gene thereof are introduced into smooth muscle cells having atherosclerosis, and activated smooth muscle cells and blood vessels are introduced. By inducing apoptosis in vascular cells such as endothelium, arteriosclerosis formation of vascular smooth muscle cells can be suppressed. The gene can be introduced into smooth muscle cells by a known method for introducing genes into eukaryotic cells. For example, DNA is mixed with a commercially available reagent such as DNA-calcium phosphate precipitation (calcium phosphate method) or ribofectamine. Lipofection, and further, a transfection method such as a retrovirus method and an electroporation method can be used.

By using this method of inhibiting arteriosclerosis of vascular smooth muscle cells to treat arteriosclerotic smooth muscle cells, gene therapy of arteriosclerotic cells can be performed.

Further, the gene of SEQ ID NO: 3, 5, or 7 in the sequence listing of the present invention induces apoptosis in activated cells, so that the gene is introduced into cancer cells to induce apoptosis. Thus, it can be used for gene therapy aimed at killing and removing cancer cells and tissues. Example 1. Cloning and identification of the F2777 (SEQ ID NO: 1) and H2A (SEQ ID NO: 3) genes

A. Culture of smooth muscle cells from plaque (P) and non-plaque (NP) sections of the aorta

Smooth muscle cell (SMC) tissue was established by the explant method. 4.5 g g 1 glucose, 10% fetal calf serum (Gibco BRL) and antibiotics (100 g / m 1 penicillin, 100 g / m 1 streptomycin sulfate) Cells were cultured using Dulbecco's Modified Eagle Medium (D-MEM) containing salt and 25 ^ g / ml amphotericin B).

B. Preparation of RNA from cultured cells

RNA was purified from the above cultured cells by the following method using TRI REAGENT (manufactured by Sigma).

(Purification method)

1. Remove the surface area of the plate medium to 10 cm ^2, remove 1 ml of Tri-Regent, and leave at room temperature for 5 minutes.

2. Dispense 1 ml each of the lysis solution into a centrifuge tube with a microphone opening for 5 ml. 3. To the lysate dispensed in 1 ml portions, add 0.2 ml of black-mouthed form and mix vigorously for 15 seconds, then leave at room temperature for 2 to 15 minutes.

4. Centrifuge for 15 minutes at 1200 xg at 4 ° C.

5. For each tube of lysate, transfer the colorless aqueous phase (top phase) to a new tube.

6. Add 0.5 ml of isopropanol, mix, and leave at room temperature for 5 to 10 minutes.

7. Pellet RNA for 10 minutes at 1200 xg at 4 ° C.

8.Wash the RNA pellet with 1 ml of 75% ethanol, and Spin with Vortex stirrer at 0 X g for 5 minutes.

9. Dry the RNA pellet and then resuspend in RNAse free water or buffer.

C. Comparison and analysis of aortic plaque (P) and non-plaque (NP) gene expression using the 'Differential Display' method

P and NP gene expression were compared and analyzed using the differential display method (SCIENCE 257, 967-971, 1992; Molecular Biotechnology 10, 261-267, 1998). The method used is shown below.

1. Using the following reaction mixture, 20 g of total RNA was treated with DNase at 37 ° C for 30 seconds.

1011-1: ^ & 36 inhibitor (Rnasin, Promega)

10 U DNase I (Promega)

10 mM Tris — HC1 buffer (pH 8.3) 50 mM KC1

1.5 mM Mg C1a

20 g total RNA

Add D EPC treated water to bring it to 501.

30 ° C, 30 minutes

2. Extract with phenol Z black mouth form.

3. Precipitate for 20 minutes at 170 ° C using Ι / l O V o l of 3 M NaAc p H 4.8 and 2.5 V o l ethanol.

4. Spin at 4 ° C for 5 minutes.

5. Resuspend in 201 D EPC treated water.

6. Denature the RNA at 65 ° C for 5 minutes and cool on ice.

7. anchored oligo one _{d T (T 1 2 NA,} T 12 NT, T 12 NG, T 12 N C) Dilute the primers to a final concentration of 50 iM.

8. Reverse transcribe (in four tubes).

100 D EPC treated water

1 1 DNA free and total RNA

4 it 1 of 5X reverse transcription buffer

2 1 0.1 M D T T

1 1 of 10 mM dNTP stock

1 1 reverse transcriptase

T _{1 2} NA, T ₁₂ NT of, T _12, NG, or T ₁₂ NC (used for each reaction)

37 ° C, 60 minutes

9. Make a PCR reaction stock (1001 total)

1 5 1 ³² P-d AT P

5 0 ^ 1 1 0 XPCR buffer

5 n 1 0.2 mM d NT P

10 1 T a q Polymerase (5 UZ 1)

2 0 1 H 2 O

1 0. Each RNA sample, the same T ₂ NAT ₁₂ NT as that used for the reverse transcription, T _{1 2} NG, or T丄₂ NC 4 kinds of anchor one Origo d T primer (each RN A sample 4 Tube).

2 1 RT—PCR product

1 2 I RCR reaction stock

4 5 z 1 H ₂ O

3 a 1 Origo d T plus primer one in each tube _{_{(T 12 NA, T 1 2}} NT, T 12 NG, or T ₁₂ NC)

1 1. Transfer the solution contained in each tube to a small Eppendorf tube. (Eppendorf) Divide into three (191 each), add 11 random primers to each tube (in this case, three different random 'primers) and add each tube Add a drop of mineral oil to the mixture.

1 2. PCR cycle

94 ° C, 5 minutes, link to the following 3 steps, 40 cycles 94 ° C, 30 seconds

40 ° C, 2 minutes

7 2 ° C, 30 seconds

7 Link at 2 ° C for 5 minutes, keep at 4 ° C

1 Prepare a 3.6% sequential gel.

30 g urea

6ml 5 XTBE buffer

9 m 1 3 8: 2 acrylic amide

2 3 m 1 water, gently mixed

120 n1 20% ASP with 601 TEMED added

14. To each PCR reaction tube, add 5 liters of mouthing buffer (same as used in sequential gels). 1 5. Denature the reaction at 95 ° C for 3 minutes.

Pre-run the gel for 20 minutes at 1300 V using 16.1 XTBE buffer.

1 7. Place samples in parallel between corresponding samples.

1 8. Electrophorese the gel in a gradient gel at a voltage of 1100 V and a voltage of 1300 V for 4-5 hours.

1 9. Dry the gel with a gel dryer for 1 hour. 2 0. — 晚 Exposure to X-ray film.

In the above comparison and analysis, X-ray photographs showing the results of comparing the gene expression of plaque (P) and non-plaque (NP) cultured smooth muscle cells derived from three ApoE-deficient mice (# 1-3) were obtained. Figure 1 shows. It should be noted that J212 in the figure means type VIII collagen.

D. Isolation and amplification of differentially expressed DNA

The following method was used to isolate and amplify DNA of a gene specifically expressed in arteriosclerotic smooth muscle cells.

(DNA isolation and amplification methods)

1. Cut out the band whose expression fluctuates from the gel, put in 50 // 1 H ₂ 〇 for 1 hour with 37, and boil for 5 minutes.

2. Secondary PCR

4 0 ^ 1 water

1 1 DNA fraction separated from gel

2 i \ 2 mM d NT P

1 fi 1 Oligo — T ₁₂ NX Primer (corresponds to primary reaction) 1 n 1 Random Primer (corresponds to primary reaction)

5 a 1 1 0 XP CR buffer—

Add a drop of mineral oil.

3. Perform PCR cycling as in (# 12) above.

4. Electrophorese the sample in 2% agarose gel in TBE buffer.

E. Purification of specifically expressed DNA

The following method was used to purify DNA, a gene that is specifically expressed in arteriosclerotic smooth muscle cells.

(Purification method using QI Aquick gel extraction kit (QIAGEN)) 1. Cut the DNA band from the agarose gel with a force razor, and place it in a 1.5-ml Eppendorf tube (manufactured by Eppendori) and weigh it.

2. Add 3 volumes of buffer-QG of gel (lOOmg ~: LOO ^ l).

3. Incubate at 50 ° C for 10 minutes (until the agarose is completely dissolved). If the color of the solution is purple, add 10 x 1 of 3 M sodium acetate.

4. Add 1 gel volume of isopropanol and mix.

5. Place the column in the collection tube, inject the sample into the column, and centrifuge at high speed for another minute.

6. Discard the flow-through and place the column in the collection tube again, inject 0.5 ml of QX 1 and spin for 1 minute.

7. Wash the column with 0.75 ml of buffer PE (at high speed for 1 minute). 8. Discard the flow-through and re-attach the column to the collection tube and spin again for 1 minute.

9. Place the column in a 1.5 ml Eppendorf tube.

Elute the DNA with 10.5 / X1 sterile distilled water (leave for 1 minute and spin for 1 minute).

1 1. Check fragments on agarose gel.

F. Cloning of differentially expressed DNA into pGEM-T vector

The differentially expressed DNA was cloned into the pGEMT vector by the following method.

(p G EM—T Vector System (Promega))

1. Prepare for the ligation reaction as follows. 2 X Rabbit 'Rigge''N''offer: 5a1p GEM—T vector: 1 1

T4DNA Ligase: 1 1

PCR product: 3 1

2 Mix and incubate at 4 ° C.

Transformation is performed using 3 JM109 competent cells.

G Sequencing

The DNA sequence was analyzed using the dideoxy termination method using fluorescent labeling.

Regarding the analysis results of the DNA sequence of the gene obtained in the present invention, the nucleotide sequence of F277 is shown in SEQ ID NO: 1 in the sequence list, and the nucleotide sequence of H2A is shown in SEQ ID NO: 3 in the sequence table. The peptide sequences in the coding region of the gene are shown in SEQ ID NOs: 2 (F277) and 4 (H2A) in the sequence listing, respectively.

H. 5 'RAC E

In order to extend the differentially expressed DNA to the 5 'end, 5' RACE was performed using the 5 'RACE (Rapid Amplification of cDNA Ends) system (Gibco BRL) (Proc. Natl. Acad. Sci. USA, 85, 8998-9002, 1988).

The experiments were performed according to the 5 'RACE protocol (5' RACE Instruction Manual).

I. c DNA library P C R

(Mouse kidney cDNA (Takara))

To clone all cDNAs of the differentially expressed genes, PCR was performed using a mouse kidney cDNA library. Forward and reverse primers were designed from the vector for constructing the library and the nucleotide sequence of the gene involved.

[Homologous search for F277 (SEQ ID NO: 1) and H2A (SEQ ID NO: 3) genes]

In the present invention, a BLAST homology search was performed on the acquired and identified genes. The results of a homology search performed on clone F277 are shown in FIG. 2, and the results performed on clone H2A are shown in FIG.

As a result of homology search, it was confirmed that clone F277 was a completely novel gene for which no similar gene was reported. Clone H2A was confirmed by a "normal" mouse whole gene database search to be a novel gene in which 34 amino acids at the C-terminal side were completely different from the homologous gene that was hit.

Example 2. Cloning and identification of “AK 0 0 2 8 7 3” (SEQ ID NO: 5 in the sequence listing) and “NM— 0 0 3 2 3 7 4” (SEQ ID NO: 7 in the sequence listing) From the human and mouse heart cDNA libraries, a gene homologous to the gene of Example 1 expressed in the cells and expressed in arteriosclerosis was cloned and identified in the same manner as in Example 1. One of the genes is "AK002873" (SEQ ID NO: 5 in the Sequence Listing), and the other is "NM-0.332374" (SEQ ID NO: 7 in the Sequence Listing). ) And the structure of the gene is shown in the same Sequence Listing. The AK 0 2 8 7 3 (from a healthy mouse) and NM— 0 3 2 3 74 (from a healthy human) gene obtained in this example and the H 2 A (arteriosclerosis-inducing mouse) obtained in Example 1 Figure 4 shows a comparison of the amino acid sequences encoded by the genes.

As is evident from FIG. 4, the gene (H 2 A) expressed in atherosclerosis and the gene of normal vascular smooth muscle cells are the 21st R from the C-terminal. (A rg) (in frame) Significantly different downstream. Therefore, specific amplification of these genes using PCR can be performed by using a pair of upstream and downstream primers of the R (A rg). Example 3. Analysis of function of atherosclerosis-expressing gene

[Construction of arteriosclerosis expression gene (H2A) expression vector]

PcDNA-H2A was prepared using the PCR method to facilitate cloning. Oligonucleotide primers were designed to include a translation initiation site and a termination codon based on the H2A sequence (H2A sense primer: 5'-ATGGCGGCTTTGCGGCCCGGAAGC_3 ', antisense primer: 5'-CACTTCCACATCGCACTGTTCATC-3 '). To create specific Kpn I and Xho I cloning sites, a Kpn I site was synthesized at the 5 'end of the sense primer and an Xho I site was synthesized at the 5' end of the antisense primer. Include 0.5 M sense and antisense primers, 200 M each dNTP, and 2.5 U Pyrobest DNA polymerase (Takara Shuzo). lng was amplified. The amplification reaction was carried out using a band thermocycler (Perkin-Elmer Cetus Instruments). After digesting the PCR product with Kpn I and Xho I and purifying it with QIA quick Gel Extraction kit (manufactured by Qiagen), the DNA fragment was digested into the Kpn I and Xho I sites of PCDNA3.1 (+) (manufactured by Invitrogen). To clone pcDNA-H2A.

[Transfection to cultured smooth muscle cells]

Transfection to mouse vascular smooth muscle cells (VSMC) was performed using the cationic lipid lipofectamine (Gibco-BRL) according to the method described in the instruction manual. For analysis by microscope, 2 Ueru Lab-Tek Chen Bakaba (manufactured by Nalge Nunc Co.) one glass per sheet, the 1 X 1 0 ⁴ cells, 1 / g of plasmid D NA and 4 L of Ribofeku Tamin Each che Transfection was performed 24 hours later using each member.

[Morphological analysis and nuclear staining of apoptotic cells]

To perform morphological analysis of the nucleus, V SMC was cultured using a tissue culture chamber slide (manufactured by Nalge Nunc). To analyze fragmented apoptotic nuclei, fix VSMC with methanol / acetic acid = 3: 1 (V / v) and stain with fluorescent dye (Hoechst 33258, 10 / xmo1 / L). did. The photograph was taken with a Nikon EFD2 fluorescence microscope (Fig. 5).

Morphological analysis of the transfected cell cultures revealed no change in cell viability in the control (Figure 5A), but cell death occurred in transfected cells cultured with pcDNA-H2A. Natsuta (Fig. 5B). Although no chromosomal staining was observed in the control (Fig. 5 (:, E), (in the cells transfected with pcDNA-H2A), the apoptotic nucleus with uniformly stained chromosomes was observed. since it had spread throughout, it has been lost normal nuclear structure and properties of apoptosis was instigation shown (Figure 5 D, F) expressed from ₀ These results H 2 a vascular smooth muscle fine Vascular smooth muscle cells are activated during the onset of atherosclerosis and begin to proliferate, migrate and produce extracellular matrix, but H 2 A It is thought that it induces apoptosis in vascular smooth muscle cells and suppresses atherosclerosis formation Gene expression of H2A in atherosclerotic blood vessels can suppress the progression of atherosclerosis Industrial availability

Stroke and heart disease, which are the 2nd and 3rd leading causes of death in Japan, are diseases caused by atherosclerosis. Therefore, preventing the development and progression of arteriosclerosis is one of the most important clinical issues. In the present invention, After culturing the respective smooth muscle cells from the lesion of pulse sclerosis and normal blood vessels and comparing the gene expression using differential 'display, the two types of increased expression in the lesion of arteriosclerosis A novel gene and a gene homologous to the gene and expressed in normal smooth muscle cells were obtained. By obtaining and identifying this gene, it becomes possible to measure the expression of this gene, and it is possible to diagnose arteriosclerosis and to use this assay to prevent arteriosclerosis and screen therapeutic agents. Was. Further, according to the present invention, arteriosclerosis can be prevented and treated by suppressing the expression of these genes which are expressed in a lesion of arteriosclerosis and are involved in the disease state. Further, in the present invention, it has been confirmed that the gene of the present invention expressed in arteriosclerosis induces apoptosis in vascular cells, and the gene is expressed in arteriosclerosis-induced smoothing. It can be introduced into muscle cells to suppress arteriosclerosis. Therefore, gene therapy can be performed by the method.

Claims

The scope of the claims

1. A DNA comprising a base sequence represented by SEQ ID NO: 1 in the sequence listing, its complementary sequence, or a sequence containing a part or all of these sequences.

2. A DNA that hybridizes with the DNA according to claim 1 under stringent conditions, and encodes a polypeptide expressed in arteriosclerosis.

3. DNA encoding the following polypeptide (a) or (b).

(a) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2 of the Sequence Listing (b) one or several amino acids were deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2 of the Sequence Listing A polypeptide consisting of an amino acid sequence and expressed in arteriosclerosis.

4. A DNA comprising a base sequence represented by SEQ ID NO: 3 in the sequence listing, a complementary sequence thereof, or a sequence containing a part or all of these sequences.

5. A DNA, which hybridizes with the DNA according to claim 4 under stringent conditions and encodes a protein expressed in arteriosclerosis.

6. DNA encoding the following polypeptide (a) or (b).

(a) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 4 in the sequence listing

(b) in the amino acid sequence represented by SEQ ID NO: 4 in the sequence listing, one or several amino acids are deleted, substituted or added, and the polypeptide is expressed in arteriosclerosis.

7. The nucleotide sequence of a gene encoding a polypeptide that induces apoptosis in vascular cells, as shown in SEQ ID NO: 5 in the sequence listing, or its complementary sequence. DNA comprising a sequence or a sequence containing a part or all of these sequences.

8. The polypeptide of SEQ ID NO: 2 which hybridizes with the DNA of claim 7 under stringent conditions, is expressed in normal smooth muscle cells, and is expressed in arteriosclerosis. And a gene encoding a polypeptide that induces apoptosis in vascular cells.

9. A gene encoding the following polypeptide (c) or (d).

(c) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 6 in the sequence listing (d) In the amino acid sequence shown in SEQ ID NO: 6 in the sequence listing, one or several amino acids were deleted, substituted or added Consists of an amino acid sequence, is expressed in normal smooth muscle cells, and is expressed in arteriosclerosis.Homologous to the polypeptide of SEQ ID NO: 2 in the sequence listing, and further induces apoptosis in vascular cells. Polypeptide.

10. Contains the nucleotide sequence of a gene encoding a polypeptide that induces apoptosis in vascular cells, or its complementary sequence, or a part or all of these sequences, as shown in SEQ ID NO: 7 in the sequence listing. DNA characterized by consisting of a sequence.

11. It hybridizes with the DNA according to claim 10 under stringent conditions, has homology to the polypeptide of SEQ ID NO: 2 in the sequence listing expressed in arteriosclerosis, and further has vascular cells. A gene that encodes a polypeptide that induces apoptosis.

12. A gene encoding the following polypeptide (c) or (d).

(c) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 8 in the sequence listing; (d) one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 8 in the sequence listing Consisting of the amino acid sequence A polypeptide that is expressed in normal smooth muscle cells and expressed in arteriosclerosis, has homology to the polypeptide of SEQ ID NO: 2 in the sequence listing, and further induces apoptosis in vascular cells.

13. The base according to claim 1, 4, 7, or 10, which hybridizes with the base sequence according to claim 1, 4, 7, or 10 under stringent conditions, and in arterial smooth muscle cells. An antisense oligonucleotide that suppresses the expression of a sequence gene.

14. A polypeptide comprising an amino acid sequence represented by SEQ ID NO: 2 in the sequence listing.

15. The amino acid sequence shown in SEQ ID NO: 2 in the sequence listing has an amino acid sequence in which one or several amino acids have been deleted, substituted or added, and is similar to a protein expressed in arteriosclerosis. Polypeptide having a structure.

16. A polypeptide comprising an amino acid sequence represented by SEQ ID NO: 4 in the sequence listing.

17. The amino acid sequence shown in SEQ ID NO: 4 has one or several amino acids deleted, substituted or added, and is similar to the protein expressed in arteriosclerosis. Polybeptide having a structure.

18. A polypeptide comprising an amino acid sequence represented by SEQ ID NO: 6 in the sequence listing and inducing apoptosis in vascular cells.

1 9. The amino acid sequence shown in SEQ ID NO: 6 has one or several amino acids deleted, substituted or added, expressed in normal smooth muscle cells, and has atherosclerosis. Polypeptide having homology to the polypeptide of SEQ ID NO: 2 expressed in the disease list and having a similar structure to the polypeptide that induces apoptosis in vascular cells De.

20. A polypeptide comprising an amino acid sequence represented by SEQ ID NO: 8 in the sequence listing and inducing apoptosis in vascular cells.

21. One or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 8 in the sequence listing, and are expressed in normal smooth muscle cells and have atherosclerosis A polypeptide having homology to the polypeptide of SEQ ID NO: 2 in the sequence listing expressed in the disease, and having a similar structure to that of the polypeptide that induces apoptosis in vascular cells.

22. An antibody, which is induced by using the polypeptide according to any one of claims 14 to 21 and specifically binds to the polypeptide.

23. The antibody according to claim 22, wherein the antibody is a monoclonal antibody.

24. The antibody according to claim 22, wherein the antibody is a polyclonal antibody.

25. A probe for diagnosis of arteriosclerosis having a nucleotide sequence that hybridizes to the nucleotide sequence according to claim 1, 4, 7, or 10 under stringent conditions.

26. A nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence according to claim 1, 4, 7, or 10 is an antisense strand of the nucleotide sequence according to claim 1, 4, 7, or 10. 26. The probe for diagnosing arteriosclerosis according to claim 25, which comprises all or part of

27. A microarray or a DNA chip for diagnosing arteriosclerosis, wherein at least one of the DNAs according to claim 25 or 26 is immobilized.

28. The antibody according to any one of claims 22 to 24 and / or claim 25 or claim 25. A diagnostic agent for arteriosclerosis, comprising the diagnostic probe according to 26.

2 9. A new kit for diagnosis of arteriosclerosis, comprising the antibody according to any one of claims 22 to 24 and Z or the diagnostic probe according to claim 25 or 26.

30. A method for diagnosing arteriosclerosis, comprising obtaining a sample from a test tissue, and measuring the expression of the gene according to claim 1, 4, 7, or 10 in the sample.

31. A method for diagnosing arteriosclerosis, wherein the measurement of the expression of the gene according to claim 30 comprises the use of quantitative PCR.

3 2. As PCR primers, primers of SEQ ID No. 9 (Forward) of Sequence Listing and SEQ ID No. 10 (Reverse), and Z or Sequence No. 11 (Forward) of Sequence Listing and Sequence of Sequence Listing 31. The method for diagnosing arteriosclerosis according to claim 31, wherein a primer of No. 12 (Reverse) is used.

3 3. An artery characterized in that the expression of the gene according to any one of claims 30 to 32 is measured using a microarray method, a DNA chip method, an RT_PCR method, or a Northern blotting method. How to diagnose sclerosis.

3 4. Obtain a sample from the test tissue, culture it, and measure one or more of the polypeptides described in SEQ ID NOs: 2, 4, 6, and 8 in the sequence listing generated by gene expression in the sample. A method for diagnosing arteriosclerosis,

35. The method for diagnosing arteriosclerosis according to claim 34, wherein the antibody according to any one of claims 22 to 24 is used.

3 6. A test substance is administered to an arteriosclerosis-onset animal model, and the expression of one or more genes having the nucleotide sequences described in SEQ ID NOs: 1, 4, 7, and 10 in the sequence listing, and Z or one or more of the polypeptides set forth in SEQ ID NOs: 2, 4, 6, and 8 in the sequence listing, or partial polypeptides thereof A method for screening a drug for preventing or treating arteriosclerosis, which comprises measuring the production of a drug.

37. The method for screening for a drug for preventing or treating arteriosclerosis according to claim 36, wherein the atherosclerosis onset model animal is an ApoE-deficient mouse.

38. The antisense oligonucleotide according to claim 13 is introduced into arterial smooth muscle cells, and expression of the gene comprising the nucleotide sequence according to claim 1, 4, 7, or 10 in arterial smooth muscle cells is induced. A method for suppressing the expression of an atherosclerosis-onset gene, which comprises suppressing the expression.

39. A vascular smooth muscle cell characterized by introducing the DNA gene according to any one of claims 4 to 12 into a smooth muscle cell having atherosclerosis and inducing apoptosis in a vascular cell. Arteriosclerosis formation suppression method.

40. A method for gene therapy of atherosclerotic cells, which comprises treating the arteriosclerosis-induced smooth muscle cells using the method for suppressing arteriosclerosis formation of vascular smooth muscle cells according to claim 39. .