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WO2006098304A1 - Nouvelle proteine secretoire derivee d’une cellule pituitaire et utilisation de ladite proteine - Google Patents

Nouvelle proteine secretoire derivee d’une cellule pituitaire et utilisation de ladite proteine Download PDF

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Publication number
WO2006098304A1
WO2006098304A1 PCT/JP2006/304969 JP2006304969W WO2006098304A1 WO 2006098304 A1 WO2006098304 A1 WO 2006098304A1 JP 2006304969 W JP2006304969 W JP 2006304969W WO 2006098304 A1 WO2006098304 A1 WO 2006098304A1
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seq
polypeptide
amino acid
polynucleotide
acid sequence
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Japanese (ja)
Inventor
Kinji Inoue
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Saitama University NUC
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Saitama University NUC
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/711—Natural deoxyribonucleic acids, i.e. containing only 2'-deoxyriboses attached to adenine, guanine, cytosine or thymine and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/06—Drugs for disorders of the endocrine system of the anterior pituitary hormones, e.g. TSH, ACTH, FSH, LH, PRL, GH
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/02—Non-specific cardiovascular stimulants, e.g. drugs for syncope, antihypotensives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/04—Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/12—Antihypertensives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • the present invention relates to a composition for promoting cell growth, maintaining survival, or suppressing tumor growth. More specifically, the present invention relates to a composition comprising a polypeptide involved in promoting cell growth and maintaining survival, a nucleotide encoding the polypeptide, an antagonist to the polypeptide, or the like.
  • Control of cell proliferation and maintenance of survival are considered to be extremely important mechanisms for maintaining biological order. For example, if cell growth control is not fully functioning and disordered cell growth can occur, it can cause serious diseases such as cancer, while cells are properly grown or maintained. If not, the onset of a disease related to the developmental disorder of the in vivo organ or the living body itself is expected.
  • cancer remains the leading cause of death due to disease, and the development of drugs and treatment methods for the effective treatment of cancer is the most important in the medical and biological fields. It is a research subject.
  • the pituitary gland is one of the organs deeply involved in functions such as growth, reproduction, and immunity.
  • the pituitary gland is an organ that secretes hormones. Factors expressed and functioning in this organ are expected to play an important role in the control of biological growth. In view of the function of the pituitary gland at the cellular level, the pituitary gland seems to play some regulatory role in the growth, proliferation, and maintenance of cells that form various biological organs.
  • Patent Document 3 it has been reported that the gene expression rate in gastric cancer is high (Patent Document 3), and more recently, the cgrl l gene has been identified as a gene suggested to be related to pain (Patent Document). 4) There is no unified view of the actual function of cgrl l, and the function has not yet been elucidated. In addition, it depends on what form the protein encoded by cgrl l functions, ie, the ability to function as it is translated into protein, the ability to function as a secreted protein, or the sugar chain, etc. There is a lack of information that is important for understanding functional mechanisms in vivo, such as whether post-translational modifications are performed.
  • Non-Patent Document 1 Madden et al. Cancer Res. 56; 5384_5390 1996
  • Patent Document 1 International Publication WO99 / 01581
  • Patent Document 2 International Publication WO 97/45542
  • Patent Document 3 International Publication WO2005 / 010213
  • Patent Document 4 International Publication WO03 / 016475
  • an object of the present invention is to provide a composition for promoting cell growth or maintaining survival, and a method for treating a related disease.
  • an object of the present invention is to provide a composition for inhibiting cell growth and a method for treating a related disease.
  • the present invention also relates to a diagnostic composition for determining the presence or absence of onset of cancer, hypertension, etc.
  • the purpose is to provide products and diagnostic methods.
  • Another object of the present invention is to provide a cglrl-encoded protein that functions in an actual living body.
  • the present invention relates to a cgrl gene product as a factor that functions in the pituitary gland that plays an important role in controlling the growth of a living body, and is considered to be deeply involved in cell growth control. This is based on the finding. Specifically, focusing on differences in protein expression between pituitary cells with different hormone-producing ability, we searched for factors that are only expressed in hormone-producing cells, and identified cgrl l as one of them. did. The inventor clarified a result completely different from the conventional finding that the cgrl l gene product is involved in cell suppression downstream of p53, and focused on the novel activity of the cgrl l protein to solve this problem. It came to do.
  • the present invention relates to the following (1) to (34).
  • a first aspect of the present invention is a cell proliferation promoting or comprising the polypeptide shown in the following (a) or (b), or an agonist thereof, and a pharmaceutically acceptable carrier: A composition for maintaining survival.
  • the second aspect of the present invention is “promoting cell growth, comprising a recombinant vector comprising a polynucleotide shown in the following (a) or (b) and a pharmaceutically acceptable carrier: Or a composition for maintaining survival.
  • SEQ ID NO: 1 SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 or SEQ ID NO: 11
  • the third aspect of the present invention is “the composition according to (1) or (2) above, wherein the cells are vascular endothelial cells”.
  • the fourth aspect of the present invention is “the composition according to (1) or (2) above, wherein the cell is a cell forming a pituitary gland”.
  • the fifth aspect of the present invention is “the composition according to (1) or (2) above, wherein the cell is a neuronal cell or a chromaffin cell”.
  • the sixth aspect of the present invention is the above-mentioned (used in the treatment or prevention of a blood flow disorder-related disease (
  • the seventh aspect of the present invention is the above (1) or (1) used for the treatment or prevention of hypotension.
  • the eighth aspect of the present invention is the above-mentioned (used for the treatment or prevention of a developmental disorder-related disease (
  • a ninth aspect of the present invention is the above (1) or (9)
  • the tenth aspect of the present invention is “cell growth inhibition or survival comprising an antagonist to the polypeptide shown in the following (a) or (b) and a pharmaceutically acceptable carrier”: Composition for suppression.
  • the twelfth aspect of the present invention is “the composition according to (11) above, wherein the antibody specifically binds to a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 13 or 14”. It is.
  • the thirteenth aspect of the present invention includes "a recombinant vector containing a nucleic acid that inhibits the function of the polynucleotide shown in the following (a) or (b)” and a pharmaceutically acceptable carrier.
  • polynucleotide J which is a nucleotide and the polypeptide encoded by the polynucleotide has a cell growth promoting activity or a survival maintaining activity.
  • a fourteenth aspect of the present invention is “the composition according to (13) above, wherein the nucleic acid that inhibits the function is an antisense DNA or RNA against the cgrl l gene”.
  • the fifteenth aspect of the present invention is “the composition according to (10) or (13) above, wherein the cell is a vascular endothelial cell”.
  • a sixteenth aspect of the present invention is "the composition according to (10) or (13) above, wherein the cell is a cell forming a pituitary gland".
  • the seventeenth aspect of the present invention is the above (10) or (13
  • the eighteenth aspect of the present invention is “the pharmaceutical composition according to the above (10) or (13), which is used for the treatment or prevention of a developmental disorder-related disease”.
  • the nineteenth aspect of the present invention is “the pharmaceutical composition according to the above (10) or (13), which is used for the treatment or prevention of hypertension”.
  • a blood flow disorder-related disease, hypotension, developmental disorder-related disease, cancer comprising an antibody against the polypeptide shown in the following (a) or (b)”: High blood pressure Composition for diagnosis of presence or absence, prognosis, or grade of malignancy or neurological disease.
  • the twenty-first aspect of the present invention is the diagnostic composition according to the above (20), wherein the antibody specifically binds to a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 13 or 14. Things ".
  • the twenty-second aspect of the present invention is “an antagonist against the polypeptide shown in the following (a) or (b).
  • a twenty-third aspect of the present invention is “an antibody against a polypeptide shown in the following (a) or (b):
  • the 24th aspect of the present invention is “the antibody according to the above (23), which specifically binds to a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 13 or 14.”
  • a twenty-fifth aspect of the present invention is a method for treating a blood of a mammal comprising administering a therapeutically effective amount of a polypeptide shown in the following (a) or (b), or an agonist thereof: A method for the treatment or prevention of flow disorders-related diseases, hypotension, developmental disorder-related diseases or neurological diseases.
  • the twenty-sixth aspect of the present invention is a cancer, developmental disorder in mammals comprising administering a therapeutically effective amount of an antagonist to the polypeptide shown in the following (a) or (b): A method for treating or preventing harm-related diseases or hypertension.
  • a twenty-seventh aspect of the present invention is “the method according to (26) above, wherein the antagonist is an antibody”.
  • the twenty-eighth aspect of the present invention includes "administering a therapeutically effective amount of a recombinant vector comprising a nucleic acid that inhibits the function of the polynucleotide shown in the following (a) or (b)”
  • a method for treating or preventing cancer, developmental disorder-related disease or hypertension in mammals includes "administering a therapeutically effective amount of a recombinant vector comprising a nucleic acid that inhibits the function of the polynucleotide shown in the following (a) or (b)"
  • a twenty-ninth aspect of the present invention is “the method according to (28) above, wherein the nucleic acid that inhibits the function is an antisense DNA or RNA against the cgrl l gene”.
  • the thirtieth aspect of the present invention includes "quantitatively detecting the amount of the polypeptide shown in the following (a) or (b) in tissue cells or blood obtained from mammals" Obviously
  • Blood flow disorder-related diseases hypotension, developmental disorder-related diseases, cancer, hypertension, heart disease or neurological disease diagnostic method.
  • a thirty-first aspect of the present invention is “a method for producing an immortalized cell by introducing a recombinant vector containing a polynucleotide shown in the following (a) or (b) into a cell:
  • polynucleotide that hybridizes with a complementary strand of a polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 or SEQ ID NO: 11 under highly stringent conditions
  • SEQ ID NO: 1 SEQ ID NO: 3
  • SEQ ID NO: 5 SEQ ID NO: 7
  • SEQ ID NO: 9 SEQ ID NO: 11 under highly stringent conditions
  • a polynucleotide in which the polypeptide encoded by the polynucleotide has a cell growth-promoting activity or survival-sustaining activity.
  • a thirty-second aspect of the present invention is “the cell obtained by the method according to (31) above”.
  • a thirty-third aspect of the present invention is “a polypeptide shown in the following (a) or (b).
  • polypeptide comprising the amino acid sequence represented by SEQ ID NO: 8, SEQ ID NO: 10 or SEQ ID NO: 12
  • polypeptide in the amino acid sequence represented by SEQ ID NO: 8, SEQ ID NO: 10 or SEQ ID NO: 12, consisting of an amino acid sequence having one or several amino acid substitutions, deletions or insertions, and cell growth promoting activity or A polypeptide having a life-sustaining activity ".
  • a thirty-fourth aspect of the present invention is “the polynucleotide shown in the following (a) or (b).
  • polynucleotide that hybridizes with a complementary strand of a polynucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 7, SEQ ID NO: 9 or SEQ ID NO: 11 under highly stringent conditions, and encoded by the polynucleotide
  • SEQ ID NO: 7 the nucleotide sequence represented by SEQ ID NO: 7
  • SEQ ID NO: 9 the nucleotide sequence represented by SEQ ID NO: 9 or SEQ ID NO: 11 under highly stringent conditions
  • composition according to the present invention it is possible to develop a pharmaceutical effective for the prevention and / or treatment of cancer.
  • composition according to the present invention By using the composition according to the present invention, it becomes possible to develop a pharmaceutical effective for the prevention and / or treatment of various diseases related to blood flow disorders.
  • composition according to the present invention By using the composition according to the present invention, it becomes possible to develop a pharmaceutical effective for the prevention and Z or treatment of hypertension, hypotension and the like.
  • composition according to the present invention By using the composition according to the present invention, it becomes possible to develop a pharmaceutical effective for prevention and Z or treatment of developmental disorders.
  • composition according to the present invention By using the composition according to the present invention, it becomes possible to develop a pharmaceutical effective for prevention and Z or treatment of neurological diseases and the like.
  • composition according to the present invention By using the composition according to the present invention, it is possible to contribute to the development of products used for industrial applications such as maintaining the survival of cultured cells, or to development in agricultural applications.
  • the composition according to the present invention includes means for detecting the cgrl gene or its product, which is strongly involved in the secretory function of various cells. For example, by measuring the concentration in the blood, it becomes possible to develop a product for examining the degree of aging and fatigue of a living body.
  • FIG. 1 Two-dimensional electrophoresis using a pituitary-derived cell line produced by hormone-producing cells (Mt T / S) but not by hormone-producing cells (MtTZEB-3) The result of identifying the quality (spot2) is shown.
  • FIG. 2 shows a mass spectrometric pattern of a tryptic peptide of Spot2. This spot was found to correspond to the cgrl 1 gene product.
  • FIG. 3 shows alignment of amino acid sequences of rat and human derived cgrl 1 proteins.
  • FIG.5 Alignment of amino acid sequence of cgrl 1 protein from foot (Rattus norvegicus), mouse (Mus musculus), human (Homo sapien s), and N-terminal amino acid analysis of secreted cgrl 1 protein Shows the actual signal sequence (underlined) as revealed by R2 and H2 represent rat and human antigenic sites, respectively.
  • FIG. 6 shows the position of a hypoxic response element found in the cgrl 1 promoter region.
  • A shows the HIF-1 binding consensus sequence
  • B shows the position of the hypoxia response element present in the promoter region of the cgrl 1 gene.
  • FIG. 7 Quantitative PCR was performed on pituitary-derived hormone-producing cells (MtTZS cells) and non-hormone-producing cells (MtT / Se cells, MtTZSM cells, MtT / E_B3 cells). The result of having measured is shown.
  • FIG. 8 shows the results of immunohistochemical staining using an antibody against cglrl protein.
  • A shows staining of MtTZS cells using anti-cgrl 1 antibody and B using anti-growth hormone antibody.
  • FIG. 9 Results of immunohistochemical staining using anti-cgrl 1 antibody on the pituitary gland (A), enlarged view of the anterior pituitary gland (B), large intestine (C), and outer follicle membrane of mature follicle (D) Indicates.
  • PN, PI, and PD represent the posterior pituitary, the middle pituitary, and the anterior pituitary, respectively.
  • FIG. 10 shows a vector construct for cgrl l gene transfer.
  • FIG. ll shows the result of immunostaining using the anti-egrl protein antibody after introducing the cgrl gene into MtT / E-B3 cells (A) and MtT / Se (C). In both cases, a positive reaction was observed in the Golgi apparatus. In contrast, no positive reaction was observed in MtT / E_B3 cells (B) and MtT / Se cells (D) into which only the vector was introduced.
  • Fig. 12 Shows the results of Western blotting using anti-cgrl protein antibodies on cell extracts, culture supernatants, and cell extracts of MtT / S cells forcibly expressing cgrl 1 protein.
  • FIG. 13 Shows the results of stamp stamping using anti-cgrl 1 protein antibody using cgrl l protein non-expressing cells (lane 1) and cgrl l protein expressing cells (lane 2) as samples. . M: Marker
  • FIG. 3 is a diagram showing that a blood vessel-rich tumor was formed at the transplantation site.
  • FIG. 16 shows a graph comparing the growth rates of cells in which the cglrl expression vector was forcibly expressed (MtT / E-B3-So) and cells into which only the vector was introduced (MtT / E-B3-Ve).
  • cgrl l forced expression PC12 cells (clone C to G) and cgrl l not forcibly expressed PC12 cells (control) were cultured in serum-free medium, and the number of viable cells on day 8 was compared. The graph is shown.
  • FIG. 19 Viable cells cultured in MtT / Se cells (cglrl non-expressing, estrogen-dependent growth) with medium containing CGR11 (serum-free), medium containing serum, and medium (serum-free) Is a graph of the number of.
  • FIG. 20 shows changes in blood pressure after injecting purified secreted cgrl into the abdominal cavity of rats. The arrow indicates the time when cgrl l protein was injected.
  • the blood pressure is shown as an average value of dilated blood pressure and systolic blood pressure.
  • the cgrl polynucleotide which is one of the active ingredients of the composition of the present invention includes not only DNA consisting of the base sequence represented by SEQ ID NO: 1, 3, 5, 7, 9 or 11, but also SEQ ID NO: 1, It hybridizes under high stringent conditions with DNA consisting of a base sequence complementary to DNA consisting of the base sequence represented by 3, 5, 7, 9 or 11 and has cell growth promoting activity or survival maintenance activity. Also included are those consisting of DNA encoding a polypeptide.
  • the “cell” may be any cell as long as the cgrl polypeptide can act, for example, animal cell, preferably human, rat, mouse, urushi, horse, hidge. , Cells from chickens, birds, cats, etc.
  • the polynucleotide of the present invention also includes the “gene” of the present invention.
  • the gene of the present invention includes cDNA and genomic DNA, and includes not only exons and introns of the gene, but also transcriptional regulatory regions such as a promoter and an enzyme.
  • the polynucleotide of the present invention includes DNA and RNA, and may be double-stranded or single-stranded. In the case of double-stranded, double-stranded DNA, double-stranded RNA or DNA and It may be a hybrid with RNA.
  • DNA that can hybridize with the polynucleotide containing the nucleotide sequence represented by SEQ ID NO: 1, 3, 5, 7, 9, or 11 under stringent conditions includes SEQ ID NO: 1, 3, 5, 7, 9, or Preferably about 70% or more, more preferably about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90% , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, most preferably about 99% of polynucleotides that contain a homologous nucleotide sequence. It is done.
  • the stringent condition is a hybridization condition that is easily determined by those skilled in the art, and is generally an empirical calculation that depends on the probe length, the washing temperature, and the salt concentration. In general, the longer the probe, the higher the temperature for proper annealing. The temperature decreases as the temperature increases and the probe becomes shorter. However, hybrid formation generally depends on the ability of the denatured DNA to reanneal when the complementary strand is present in an environment near its melting point but below it.
  • the washing of the post-hybridization of the final letter should be performed at a temperature of 37 ° C to 42 ° C, 0.1 X
  • a temperature of 37 ° C to 42 ° C 0.1 X
  • washing in SSC 0.1% SDS solution.
  • highly stringent conditions include washing in 65 ° C, 5 X SSC and 0.1% SDS in the washing step.
  • the cgrl polynucleotide can be obtained from, for example, a cell derived from the pituitary gland and producing hormone. For example, extract all proteins present in hormone-producing cells and non-hormone-producing cells as controls, and perform two-dimensional electrophoresis, etc., and compare their migration profiles, and only in hormone-producing cells. Amino acid sequence information is obtained from confirmed protein spots using mass spectrometry. Next, based on the obtained amino acid sequence information, DNA having a cgrl polynucleotide sequence can be isolated from an appropriate cDNA library or the like.
  • sequence information of a known cgrl l gene from a database or the like and obtain a cgrl l gene derived from a desired tissue or animal species based on the sequence information.
  • Cloning of the cgrl gene can be carried out based on ordinary knowledge in the art.
  • the gene can be obtained by preparing a cDNA library from cells expressing the gene and using a conventional screening method.
  • RNA is prepared from cells expressing the gene
  • cDNA is synthesized by reverse transcriptase
  • PCR primers are prepared based on the gene sequence to amplify the cDNA.
  • the cDNA may be obtained.
  • the cell that can be used for obtaining the cgrl polynucleotide in the present invention is any cell that can express any mRNA that expresses cgrl polypeptide mRNA.
  • Pituitary hormone-producing cells are preferred.
  • the movement from which the cells are derived The product may be any animal (for example, human, rat, etc.) as long as it has the gene of the present invention.
  • cgrl l To obtain mRNA for obtaining polynucleotides and cDNA, use commercially available kits such as mRNA Purification Kit (Pnarmasia) and AMV Reverse Transcriptase First-strand cDNA Synthesis Kit (Seikagaku Corporation). Use it.
  • the cgrl polypeptide in the present invention is a polypeptide comprising the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 10 or 12.
  • the “polypeptide containing substantially the same amino acid sequence” means about 60% or more, preferably about 70% or more, of the amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 10, or 12. More preferably, about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, most preferably a polypeptide having an amino acid sequence having about 99% amino acid identity and having cell growth promotion or activity.
  • the “cell” may be any cell as long as it can act on cgrl polypeptide, for example, animal cell, preferably human, rat, mouse, urushi, horse, It is a cell derived from Hedge, chicken, dog, cat, etc.
  • polypeptide and “protein” are used in the same meaning unless otherwise specified.
  • polypeptide comprising an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 10, or 12, SEQ ID NO: 2, 4, 6, 8, 10 Or one or several (preferably:! To about 30, more preferably about 1 to 10, more preferably :! to 5) amino acids in the amino acid sequence represented by 12 are deleted, It is a polypeptide having an amino acid sequence substituted or added and having cell growth promotion or activity.
  • the “cell” may be any cell as long as the cglrl polypeptide can act, for example, an animal cell, preferably a human, rat, mouse, urushi, horse, hidge. It is a cell derived from nitrite, nu, cat, etc.
  • amino acid deletions, additions and substitutions are present in isolated natural polypeptides.
  • the substitution of a specific amino acid residue is a known method such as Guppedduplex method or Kunkel method using a commercially available kit (for example, MutanTM-G (TAKARA), MutanTM-K (TAKARA)).
  • TAKARA MutanTM-G
  • TAKARA MutanTM-K
  • the C-terminus of the cgrl polypeptide in the present invention is usually a carboxyl group (_C0OH) or a carboxylate (_C00_), and the carboxyl group is an amide (one CONH2). Or may be chemically modified with ester (_C0OR).
  • C1-6 alkyl group for example, methinole, ethyl, n-propyl, isopropyl, n-butyl
  • C3-8 cycloalkyl group for example, cyclopentyl, cyclohexylene
  • C1-6 aryl group for example, phenylolene, mononaphthyl
  • phenyl-1-C1-2anolyl group for example, benzyl, phenethyl
  • ⁇ -naphthyl-C1-2 alkyl group for example, a naphthylmethyl
  • the polypeptide of the present invention has a carboxyl group in the polypeptide chain other than at the C-terminus, the polypeptide of the present invention includes those in which the carboxyl group is amidated or esterified. Examples of the ester in this case include the above-mentioned esters.
  • the N-terminus of the polypeptide of the present invention is usually an amino group (one NH2), but the amino group may be chemically modified with a C16 acyl group such as a formyl group or a acetyl group. .
  • the daltamyl group generated by cleaving the N-terminal side in vivo is pyrologretamine oxidized, or a substituent on the side chain of an amino acid in the molecule (for example, OH, —SH, amino group, imidazole group, indole group, Polypeptides of the present invention include those in which a guanidino group or the like) is chemically modified with an appropriate functional group (for example, formyl group, acetyl), or a sugar chain.
  • a peptide containing a partial amino acid sequence in the cglrl polypeptide of the present invention may be included in the composition of the present invention.
  • the partial peptide can also be an antigen for preparing the antibody of the present invention. That is, the partial peptide of the present invention is the same or substantially the same as the amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6. As long as it contains a part of the amino acid sequence of the same amino acid sequence.
  • the number of amino acids constituting the partial peptide of the present invention is at least 10 or more, preferably 30 or more, more preferably 80 or more.
  • the partial peptide of the present invention may be mechanically modified such that the C-terminus is a carboxyl group (_COH) and the N-terminus is an amino group (_NH2).
  • the cgrl l polypeptide or a salt thereof in the present invention is obtained from a cultured cell or tissue derived from a human animal (eg, rat, mouse, etc.) expressing the cgrl l polypeptide by a conventional technique in the art. Extraction and isolation from the culture can be performed by culturing a transformant that can be extracted or isolated, or can be expressed in a state in which the DNA encoding the polypeptide of the present invention can be expressed as described later. Can also be prepared. When preparing from human tissues or cells, homogenize human tissues or cells and extract with acid, etc., and use the resulting extract for hydrophobic chromatography, reverse phase chromatography, ion exchange chromatography, etc. Isolation and purification can be achieved by combining these various chromatographies.
  • the partial peptide or its salt in the present invention is produced by a known peptide synthesis method or cgrrl polypeptide cleaved with an appropriate peptidase (eg, trypsin, chymotrypsin, arginyl endopeptidase). be able to.
  • an appropriate peptidase eg, trypsin, chymotrypsin, arginyl endopeptidase.
  • the peptide synthesis method for example, either a solid phase synthesis method or a liquid phase synthesis method may be used. That is, the desired peptide is produced by condensing the partial peptide or amino acid that can constitute the polypeptide of the present invention and the remaining part, and removing the protecting group when the product has a protecting group.
  • the partial peptide of the present invention can be isolated and purified by combining ordinary purification methods such as solvent extraction, distillation, column chromatography, high performance liquid chromatography, recrystallization and the like.
  • cgrl polypeptide obtained by the above method or a partial peptide thereof is a free form
  • it can be converted to an appropriate salt by a known method.
  • the free form is obtained by a known method.
  • a recombinant vector incorporating the cgrl 1 polynucleotide or a part thereof can be obtained by ligating the cgrl polynucleotide or a part thereof to an appropriate vector.
  • the recombinant vector is not particularly limited as long as it can be replicated in a host when it is used for cloning.
  • a vector for expressing cgrl 1 polypeptide or a part thereof a vector capable of replicating in a host and having a promoter capable of expressing a DNA fragment encoding the polypeptide, etc. Can be used.
  • Plasmid DNA includes plasmids derived from E. coli (eg, pBR322, pBR325, pUC118, pUC119, pUC18, pUC19, pCBD-C, etc.), plasmids derived from Bacillus subtilis (eg, pUB110, pTP5, pC194, etc.), and yeast-derived plasmids (eg, ⁇ 13, ⁇ 24, YCp50, YIp30, etc.), and phage DNA includes ⁇ phage.
  • animal viruses such as retrovirus and vaccinia virus, and insect virus vectors such as baculovirus and toga virus can also be used.
  • the promoter used in the present invention is not particularly limited as long as it is an appropriate promoter corresponding to the host used for gene expression.
  • SRa promoter when an animal cell is used as a host, SRa promoter, CMV promoter, SV40 promoter, LTR promoter, HSV-TK promoter, EF-1 ⁇ promoter and the like can be mentioned.
  • the host is Escherichia coli, tac promoter, ⁇ ⁇ ⁇ ⁇ promoter, lac promoter, recA promoter, e.g. PL promoter, lpp promoter, etc. If the host is Bacillus subtilis, SPOl promoter, SP02 promoter, penP promoter Etc.
  • PH05 promoter When the host is yeast, PH05 promoter, PGK promoter, GAP promoter, ADH promoter and the like can be mentioned.
  • polyhedrin promoter When the host is an insect cell, polyhedrin promoter, P10 promoter, etc. are preferable.
  • the recombinant vector of the present invention includes a cgrl 1 polypeptide or a portion of a coding cage 1J,
  • a selection marker, terminator, enhancer, splicing signal, poly A addition signal, ribosome binding sequence (SD sequence), SV40 origin of replication (SV40ori), etc. can be linked.
  • Selection markers include, but are not limited to, hygromycin resistance marker (Hygr), dihydrofolate reductase gene (dMr), ampicillin resistance gene (Ampr), kanamycin resistance gene (Kanr), neomycin resistance gene (Neor, G418) Etc. are available.
  • an appropriate signal sequence may be added to the N-terminal side of the polypeptide of the present invention.
  • Inserting a cgrl polynucleotide or a part thereof into the above-described vector means that the cloned DNA is digested with a restriction enzyme as it is or as desired, and a linker is added to the vector DNA. It can be performed by inserting into a restriction enzyme site or a multicloning site.
  • the DNA to be ligated may have ATG as a translation initiation codon on the 5 ′ end side and TAA, TGA or TAG as a translation termination codon on the 3 ′ end side. These translation initiation codons and translation termination codons can also be added using an appropriate synthetic DNA adapter.
  • the DNA to be ligated needs to be incorporated into a vector so that the polypeptide of the present invention encoded in the DNA is expressed in the host cell.
  • a transformant of cgrl can be obtained by introducing the recombinant expression vector of the present invention into a host so that the cgrl l gene can be expressed.
  • the host is not particularly limited as long as it can express the cgrl 1 gene.
  • Escherichia coli and other genus Escherichia, Bacillus subtilis and other Bacillus genus, Pseudomonas putida and other shyudomonas genus, Rhizobium meliloti and other lysovivum genus Bacteria belonging to, Saccharomyces cerevisiae, Szozoaccharomyces pombe, Pichia pastoris, monkey cells COS _ 7, Vero, Chinese hamster ovary cells (CHO cells) Or insect cells such as Sf 9 and Sf 21.
  • a method for introducing a recombinant vector into Escherichia coli a method using calcium ions (Cohen et al., 1972), an electo mouth position method (Shigekawa and Dower, 1988) and the like can be used.
  • a method for introducing a recombinant vector into yeast the Elect Mouth Position method (B ecker et al., 1990), the Suguchi Higuchi plast method (Hinnen et al., 1978), the lithium acetate method (Itoh et al., 1983), etc. can be used. .
  • Methods for introducing recombinant vectors into animal cells or animal cells include the DEAE dextran method (Lopata et al., 1984), the electopore position method, the phosphate phosphate method (Chen and Okayama, 1988), and the method using cationic lipids. (Elroy-Stein and Moss, 1990).
  • the medium can be any medium that is suitable for the desired cells to normally survive, for example, about 5 to 20%. MEM medium, DMEM medium, RPMI-1640 medium, etc. containing fetal bovine serum are used.
  • conditions suitable for the desired cells can be set for pH and culture temperature, and these conditions can be easily set by those skilled in the art. As described above, if the cgrl polypeptide is expressed in a cell in which immortalization or survival is desired, Cell immortalization or viability maintenance is achieved.
  • the cgrl l polypeptide or a partial peptide thereof contained in the composition of the present invention is obtained by culturing a transformant introduced with an expression vector of the cgrl l gene or a part thereof, and cgrl 1 polypeptide or a part thereof. Can be produced by isolating the polypeptide or a part thereof from the culture. “Culture” means any of culture supernatant, cultured cells or cultured cells, or disrupted cells or cells.
  • the medium contains a carbon source, a nitrogen source, inorganic salts and the like that can be assimilated by the microorganism. Either natural or synthetic media can be used as long as the culture can be carried out efficiently.
  • a carbon source carbohydrates such as glucose, fructose, sucrose and starch, organic acids such as acetic acid and propionic acid, and alcohols such as ethanol and propanol are used.
  • nitrogen sources include ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, ammonium salts of organic acids such as ammonium salts, or other nitrogen-containing compounds, peptone, meat extract, corn steep liquor, etc. Used.
  • inorganic salts include monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, and calcium carbonate.
  • the culture is performed under conditions suitable for the host cell.
  • a medium for culturing Escherichia coli LB medium, M9 medium and the like are preferable. Agents such as isopropyl-1-thio ⁇ D galactoside, 3-indolylacrylic acid can be added to make the promoter work efficiently if desired.
  • the culture is usually carried out at about 15 to 37 ° C for about 3 to 24 hours, and if necessary, aeration and agitation can be controlled.
  • cultivation is usually performed at about 30-40 ° C for about 6-24 hours, and if necessary, aeration or agitation is added.
  • Examples of the medium for culturing yeast include SD medium and YPD medium. It is preferable to adjust the pH of the medium to about 5-8. Incubate at about 20 to 35 ° C for about 24 to 72 hours, and if necessary, add aeration and agitation. Transformation where the host is an insect cell or insect When culturing the body, examples of the medium include Grace insect medium containing urine serum. The pH of the medium is preferably adjusted to about 6.2 to 6.4. Incubate at about 27 ° C for about 3 to 5 days, and add aeration or agitation as necessary.
  • a transformant whose host is an animal cell is cultured, for example, a MEM medium, DMEM medium, RPMI-1640 medium or the like containing about 5 to 20% urine fetal serum is used. .
  • the pH is preferably about 6-8.
  • the cells or cells are collected by a known method after culturing and suspended in an appropriate buffer solution.
  • a method of obtaining a crude extract of cgrl polypeptide or a partial peptide thereof by centrifugation or filtration after disrupting cells or cells by ultrasonic waves, lysozyme, Z, or freeze-thawing is appropriately used.
  • the buffer may contain a protein denaturant such as urea or guanidine hydrochloride, or a surfactant such as Triton X-100.
  • the cells or cells and the supernatant are separated by a method known per se, and the supernatant is collected.
  • Purification of the polypeptide of the present invention contained in the thus obtained culture supernatant or extract can be performed by appropriately combining known separation and purification methods.
  • known separation and purification methods methods using solubility such as salting-out solvent precipitation method, dialysis method, ultrafiltration method, gel filtration method, and mainly using differences in molecular weight such as SDS-PAGE.
  • Methods using charge differences such as ion exchange chromatography, methods using specific affinity such as affinity mouthmatography, and differences in hydrophobicity such as reversed-phase high-performance liquid chromatography
  • a method using a difference in isoelectric point such as a method or an isoelectric focusing method may be used.
  • Nucleic acids that inhibit the function of the cgrl l gene may inhibit the growth or survival of cells that function as the cgrl l gene is expressed by losing the function of the cgrl l gene. It becomes possible. As a result, for example, it is possible to suppress the growth of cells that have become cancerous or tumorous due to, for example, disordered overexpression of cglrl, or cells that may become cancerous or tumorous.
  • Examples of nucleic acids that inhibit gene functions include single-stranded nucleic acids such as antisense RNA or DNA and derivatives thereof, short-length or double-stranded RNA having a sequence complementary to a part of the gene region, etc. Can be mentioned.
  • Antisense acts as an effective inhibitor for the expression of the cgrl gene, which can be RNA or DNA or derivatives thereof.
  • Antisense RNAs are designed, for example, to hybridize with mRNA in vivo and inhibit translation from mRNA to cgrl polypeptide (Okano et al., 1991).
  • the DNA oligonucleotide is designed to be complementary to the transcription initiation region of the cglrl gene, for example, and as a result, inhibits the expression of the gene (Cohen, 1989).
  • antisense RNAs or DNAs can be introduced into cells so that they can function in vivo to inhibit the expression of cglrl genes or polypeptides.
  • antisense DNA for example, an oligonucleotide that binds to a position between about 10 and +10 of the target gene sequence is desirable.
  • RNAi is a phenomenon in which the target mRNA (for example, mRNA of the cglrl gene) is degraded by double-stranded RNA having a sequence complementary to the target mRNA.
  • the target mRNA for example, mRNA of the cglrl gene
  • double-stranded RNA having a sequence complementary to the target mRNA By artificially introducing double-stranded RNA using this phenomenon, the expression of the target gene can be suppressed. Therefore, it is considered to be based on a principle different from the above-described antisense method.
  • An antibody against the cgrl 1 polypeptide is capable of inhibiting the growth or survival of cells in which the cgrl 1 polypeptide is expressed and functioning by losing the activity of the cgrl 1 polynucleotide. As a result, it is possible to suppress the proliferation of cells that have become cancerous or tumorous due to, for example, disordered overexpression of cglrl, or cells that may become cancerous or tumorous.
  • the presence or absence of cancer due to overexpression of cgrl l polypeptide, or prognosis can be made using an antibody against the cgrl polypeptide.
  • the presence or absence of canceration or the prognosis of cancer can be determined by measuring the concentration of cgrl polypeptide present in blood obtained from a specimen using the antibody.
  • Antibodies against cgrl l include antibodies that specifically bind to cgrl l polypeptide, and antibody fragments such as Fab or F (ab ') thereof.
  • the “antibody” in the present invention includes a monoepitope specific antibody against the cgrl polypeptide, Polyepotope specific antibodies, single chain antibodies, and fragments thereof are included. These antibodies include, for example, monoclonal antibodies, polyclonal antibodies, humanized antibodies and the like.
  • Polyclonal antibodies can be prepared, for example, by incubating a mixture of immunogen and adjuvant against a mammalian host animal. Usually, the immunogen and / or adjuvant is injected multiple times subcutaneously or intraperitoneally into the host animal.
  • Immunogens include polypeptides of the invention and fusions with heterologous polypeptides or fragments thereof. For example, a peptide consisting of the amino acid sequence represented by SEQ ID NO: 13 or 14 is a very good immunogen.
  • adjuvants include complete Freud and monophosphoryl lipid A synthesis trehalose dicorynomycolate (MPL-TDM).
  • MPL-TDM monophosphoryl lipid A synthesis trehalose dicorynomycolate
  • KLH keyhole limpet hemocyanin
  • serum albumin serum albumin
  • thythyroglobulin thythyroglobulin
  • soybean trypsin inhibitor are used to enhance the immune response. After binding the immunogen such as a protein having immunogenicity, it may be digitized.
  • Monoclonal antibodies can be prepared using the Hypridoma method (Milstein et al. Cuello, 1983).
  • This method includes the following four steps: (i) immunizing the host animal or lymphocytes derived from the host animal, (ii) a monoclonal antibody secreting (or potentially secreting) linker. Collect spheres, (iii) fuse lymphocytes to immortalized cells, (iv) select cells that secrete the desired monoclonal antibody.
  • Immunogens also include cgrl polypeptide and fusions with heterologous polypeptides or fragments thereof.
  • an immunogen for example, a peptide consisting of the amino acid sequence represented by SEQ ID NO: 13 or 14 is a very excellent immunogen.
  • lymphocytes obtained from the host animal are fused with an immortalized cell line using a fusion agent such as polyethylene glycol to establish hyperpridoma cells (Goding, 1
  • a cartilage or mouse myeloma cell line in which rodents, rabbits, or human myeloma cells immortalized by transformation are used is used.
  • the cells are grown in a suitable medium containing one or more substrates that inhibit the growth or survival of unfused lymphocytes and immortalized cell lines.
  • Conventional techniques use parental cells that lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT). In this case, hypoxanthine, aminopterin and thymidine are added to a medium (HAT medium) that inhibits the growth of HGPRT-deficient cells and allows the growth of hypridoma.
  • HGPRT hypoxanthine guanine phosphoribosyltransferase
  • a preferred immortal cell line is the mouse myeloma line, which is available from the American Type Culture Collection (Manassas, VA). See Kozbor et al., 1984; Schook, 1987 for the production of human monoclonal antibodies by human myeloma and mouse human heteromyeloma cell lines.
  • Monoclonal antibody-secreting hyperpridoma cells can be isolated as single clones by limiting dilution and subculture (Goding, 1996). Suitable media include Dulbecco's Modified Eagle Medium, RPMI-1640, and in some cases, protein-free or serum-free medium. Hypridoma cells may also be grown in the ascites of suitable host animals.
  • Monoclonal antibodies can be obtained from medium or ascites, et al. Protein A sepharose, hydoxyxpatite chromatography, gel electrophoresis, dialysis, ammonium sulfate precipitation or affinity chromatography (Harlow and Lane, 1988; Harlow and Lane, 1999). It is isolated and purified by methods well known to those skilled in the art.
  • Monoclonal antibodies can also be produced by gene recombination techniques (US Pat. No. 4166452, 1979).
  • To identify the gene encoding the desired monoclonal antibody polypeptide from the hyperidoma cell line secreting the antibody of interest for example, using oligonucleotide probes that specifically bind to the mouse heavy and light chain antibody genes. May be.
  • the target antibody gene can be identified by determining the distribution 1J of the genes.
  • the isolated DNA fragment is transferred to an appropriate expression vector and the antibody gene is not produced by any other immunoglobulin protein, simian COS-7 cells, Chinese hamster ovary Transfect into (CHO) cells or host cells such as myeloma cells.
  • Isolated DNA fragments can be obtained, for example, by replacing coding sequences for human heavy and light chain constant domains with homologous mouse sequences (US Pat. No. 4,816,567; 1989; Morrison et al., 1987) or non- Modifications can be made by fusing the immunoglobulin coding sequence with all or part of the sequence encoding the immunoglobulin polypeptide.
  • non-immunoglobulin polypeptides can be substituted for the constant domain of an antibody or can be substituted for the constant domain of a single antigen binding site to prepare a chimeric bivalent antibody.
  • 7-3 Humanized and human antibodies
  • cgrl polypeptide antibodies include human rabbits or human antibodies.
  • Humanized forms of non-human antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (Fv, Fab, Fab ′, F (ab ′)) or other antibody antigen-binding regions that contain minimal sequences derived from non-human immunoglobulin.
  • Etc
  • human rabbit antibodies have one or more amino acid residues introduced from non-human immunoglobulin. These non-human amino acid residues are often selected from variable domains.
  • Humanized antibodies can be produced, for example, by replacing mouse CDRs or CDR (complementarity determining region) sequences with corresponding human antibody sequences (Jones et al., 1986; Riechmann et al., 1988; Verhoeyen et al. , 1988).
  • a humanized antibody is a human antibody that is substituted with a residue in a specific human-derived CDR and a residue in a CDR of a non-human species such as mouse, rat, or rabbit corresponding to the residue. That is.
  • nonhuman-derived residues may replace Fv framework residues of human immunoglobulin (Jones et al., 1986; Presta, 1992; Riechmann et al., 1988).
  • the “agonist” in the present invention is a substance that specifically binds to cgrl polypeptide, its receptor, or interacting factor (binding partner) to promote the biological activity of the polypeptide of the present invention. And a part of the above-mentioned antibody is also included in the “agonist”. Examples of non-antibody antibodies include, but are not limited to, polypeptides or fragments thereof, nucleic acids, and other low molecular weight compounds.
  • the “antagonist” in the present invention means a substance that specifically binds to cglrl polypeptide, its receptor, or an interaction factor (binding partner) and suppresses the biological activity of the polypeptide of the present invention. However, some of the above-mentioned antibodies are also included in “antagonists”.
  • Abutama is a nucleic acid molecule or peptide capable of specifically binding to a target protein and can inhibit the activity of the target protein. In addition, even if the activity cannot be inhibited, it can be used to examine the presence or absence of the protein because it can bind to the target protein. Therefore, aptamers to cgrl l polypeptide can inhibit the growth or survival of functioning cells by expressing cgrl l polypeptide by losing the activity of cgrl l polypeptide. .
  • a disease caused by overexpression of cgrl l polypeptide for example, the presence or absence of onset of cancer, or prognosis can be determined using an aptamer to cgrl l polypeptide. For example, the presence or absence of canceration or the prognosis of cancer can be determined by measuring the concentration of cgrl polypeptide present in blood obtained from a specimen using the aptamer.
  • RNA or DNA specifically binds to the cglrl polypeptide.
  • it is DNA.
  • Aptamers also include those that have been modified in the phosphate skeleton to increase in vivo stability, and are not particularly limited, but include phosphorothioate bonds, phosphorodithioate bonds, and phosphoramidothioates. A phosphate skeleton containing a bond, a phosphoramidate bond, a phosphordiamidate bond, or a methylphosphonate bond is preferred.
  • the factor that interacts with the cgrl l polypeptide is expected to have a promoting or suppressing effect on the cell growth promotion or survival maintenance activity of the cgrl l polypeptide. Therefore, by further adding the factor to the composition of the present invention, it is possible to achieve the effect according to the purpose.
  • “interacting factors” include those that can be assumed by those skilled in the art, such as proteins, peptides, and small compounds.
  • proteins include receptors for eg rl l polypeptides.
  • the cgrl l polypeptide or a part thereof and a candidate factor eg, a receptor
  • a candidate factor eg, a receptor
  • a cell that presents a candidate factor on its surface eg, a candidate
  • a cell that expresses a receptor protein on its surface, etc. the binding amount of cgrl l polypeptide or a part thereof with a candidate factor, or the cell, etc.
  • the presence or absence of interaction can be determined by detecting biological changes in the body.
  • the cgrl l polypeptide or a part thereof, or a candidate factor is labeled (for example, Fluorescent labeling, radiolabeling), contacting the two, and using the intensity of the signal obtained from the label (for example, fluorescence, radioactivity, etc.) as an index, detect the amount of binding of both based on conventional methods and common technical knowledge As a result, if it is possible to evaluate that both are strongly bound, the candidate factor can be determined to be an interacting factor.
  • labeled for example, Fluorescent labeling, radiolabeling
  • a cell expressing a candidate receptor is contacted with a cgrl polypeptide or a part thereof, and a cell-specific intracellular signal via the receptor (for example, cell When changes in intracellular Ca 2+ release, intracellular cAMP production, cell membrane potential fluctuation, intracellular protein phosphorylation, etc.) are detected, it is predicted that the candidate receptor actually functions as a receptor It becomes possible.
  • a composition comprising a cgrl polynucleotide, a polypeptide, an agonist for the polypeptide, etc. can be used for the purpose of promoting cell growth or maintaining the survival of the polynucleotide.
  • a composition comprising an antibody against a peptide, an antagonist, etc. can be used for the purpose of suppressing abnormal cell growth (eg, tumorigenesis).
  • the composition can also be used in vitro for use in cultured cells and the like.
  • the present composition includes substances for maintaining isotonicity of cells, substances for promoting the transfer of the active ingredients into cells, and cuffers, etc. Auxiliary substances are included. Any of these auxiliary substances can be used if they are available to those skilled in the art.
  • a composition comprising a cgrl polynucleotide, a polypeptide, an agonist for the polypeptide, etc. can be used for the purpose of promoting cell growth or maintaining the survival of the polynucleotide.
  • a composition comprising an antibody against a peptide, an antagonist, etc. can be used as a pharmaceutical composition because it can be used for the purpose of suppressing abnormal cell growth (eg, tumorigenesis).
  • the pharmaceutical composition can be expected to be effective in treating a blood flow disorder-related disease if, for example, vascular endothelial cells are used as a target. Also, if the pituitary-forming cells are used as a target, the pharmaceutical composition is considered to be a growth disorder. It can be expected to be effective in treating related diseases, while tumors or cancerous diseases If cells that show normal growth are used as targets, an effect can be expected in the treatment or prevention of cancer.
  • composition comprising the cglrl polynucleotide according to the present invention, a polypeptide, an agonist for the polypeptide, etc. has an effect of increasing blood pressure, and also inhibits the activity of the polynucleotide.
  • a composition comprising an nucleic acid or an antibody against the polypeptide, an antagonist or the like has an effect of lowering blood pressure, and thus can be used as a pharmaceutical composition.
  • the pharmaceutical composition is expected to be effective as, for example, an antihypertensive agent, a hypotensive agent, a hypotensive therapeutic agent, or a hypertensive therapeutic agent.
  • blood flow disorder-related disease refers to a disease caused as a result of hindering blood circulation in the whole or a part of the body or a disease causing hindrance to blood circulation.
  • arteriosclerosis inflammatory vasculitis, aneurysm, thrombophlebitis, and kidney disease are all diseases within the scope of common technical knowledge of those skilled in the art.
  • hypertension and “hypotension” include related diseases caused by abnormal blood pressure, such as heart diseases (for example, myocardial infarction, aortic stenosis, cardiac hypertrophy, hypertrophic cardiomyopathy and heart failure). included.
  • heart diseases for example, myocardial infarction, aortic stenosis, cardiac hypertrophy, hypertrophic cardiomyopathy and heart failure.
  • Cancer refers to a disease associated with abnormal growth or proliferation of cells, and is within the scope of common technical knowledge of those skilled in the art, such as adenocarcinoma, lymphoma, blastoma, melanoma, sarcoma, and leukemia. It refers to all diseases.
  • Neurological disease is to be understood in a general sense and includes all diseases caused by the degeneration or damage of nerve cells or para-neurons, eg Parkinson's disease, Alzheimer's disease, Huntington All diseases within the scope of common technical knowledge of those skilled in the art, such as illness, amyotrophic lateral sclerosis, spinocerebellar degeneration
  • “Developmental disorder” is a condition in which some abnormality is observed in physical growth. Related diseases include, but are not limited to, dwarfism, giantism, etc. .
  • the above cgrl polynucleotide can be used as a therapeutic agent in the form of a pharmaceutical composition that does not adversely affect the living body.
  • a pharmaceutical composition that does not adversely affect the living body.
  • such compositions contain a nucleic acid component.
  • a pharmaceutically acceptable carrier is included.
  • “Pharmaceutically acceptable carrier” includes solvents, dispersion media, coatings, antibacterial and antifungal agents, agents that act isotonically to delay adsorption and the like and are suitable for pharmaceutical administration. Yes (Gennaro, 2000).
  • the carrier and preferred for diluting the carrier include, but are not limited to, water, saline, finger solution, dextrose solution, human serum albumin, and the like.
  • Non-water soluble media such as ribosomes and non-volatile oils are also used.
  • certain compounds that protect or promote activity such as the above cgrl polynucleotides may be included in the composition.
  • the pharmaceutical composition according to the present invention includes intravenous, intradermal, subcutaneous, oral (for example, including inhalation), transdermal, and transmucosal administration, and is suitable for a therapeutically appropriate route of administration. It is formulated as follows. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application include, but are not limited to, sterile diluents such as water for injection, saline solution, non-volatile oil, polyethylene.
  • Preservatives such as glycol, glycerin, propylene glycol, or other synthetic solvents, benzenolic alcohol or other methylparabens, antioxidants such as ascorbic acid or sodium hydrogen sulfite, painless such as salt benzalkonium, prohydroin hydrochloride It may contain a chemical, a chelating agent such as ethylenediaminetetraacetic acid (EDTA), a buffer such as acetate, citrate, or phosphate, and an agent for osmotic pressure adjustment such as sodium chloride or dextrose.
  • EDTA ethylenediaminetetraacetic acid
  • buffer such as acetate, citrate, or phosphate
  • agent for osmotic pressure adjustment such as sodium chloride or dextrose.
  • the pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • acids or bases such as hydrochloric acid or sodium hydroxide.
  • Parenteral preparations are contained in ampoules, disposable glass or plastic syringes or multiple dose vials.
  • compositions suitable for injection include sterile aqueous solutions (dispersible) or dispersion media and sterile powders to be prepared at the time of use.
  • suitable carriers include physiological saline, bacteriostatic water, CREMOP HOR EL TM (BASF, Parsippany, NJ), or phosphate buffered saline (PBS).
  • the composition When used as an injection, the composition must be sterile and must have sufficient fluidity to be administered using a syringe.
  • the composition The product must be stable to chemical changes and corrosion during preparation and storage, and should prevent contamination from microorganisms such as bacteria and fungi.
  • the carrier can be made using a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures.
  • a coating agent such as lectin
  • the required particle size is maintained in a dispersion medium, and an appropriate fluidity is maintained by using a surfactant.
  • Various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal, can be used to prevent microbial contamination.
  • compositions such as sugar, mannitol, sorbitol, and agents that maintain isotonicity such as sodium chloride may be included in the composition.
  • Compositions that can delay adsorption include agents such as aluminum monostearate and gelatin.
  • Sterile injectable solutions are prepared by sterilizing the required ingredient alone or in combination with the other ingredients and then adding the required amount of the active compound in a suitable solvent.
  • dispersion media are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the other necessary ingredients discussed above.
  • Sterile powder preparation methods for the preparation of sterile injectable solutions include vacuum drying and lyophilization to prepare a powder containing the active ingredient and any desired ingredients derived from the sterile solution. It is.
  • Oral compositions usually include an inert diluent or a carrier that does not cause harm when taken into the body.
  • Oral compositions are, for example, contained in gelatin capsules or compressed into tablets.
  • the active compound is incorporated with excipients and used in the form of tablets, troches, or capsules.
  • the oral composition can also be prepared using a fluid carrier, and the composition in the fluid carrier is applied orally.
  • pharmaceutically compatible binding agents, and Z or adjuvant substances may be included.
  • Tablets, pills, capsules, troches and the like have the following ingredients or similar properties
  • excipients such as microcrystalline cellulose, binders such as gum arabic, tragacanth or gelatin
  • bulking agents such as alginic acid, PRIM GEL, or corn starch, such as starch or ratatose Lubricants such as magnesium stearate or STRROTES; lubricants such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin; or flavoring agents
  • the cgrl 1 polynucleotide and the like can be prepared as a sustained-release preparation such as a delivery system encapsulated in implantable tablets and microcapsules using a carrier that can prevent immediate removal from the body.
  • a sustained-release preparation such as a delivery system encapsulated in implantable tablets and microcapsules using a carrier that can prevent immediate removal from the body.
  • Biodegradable, biocompatible polymers can be used, such as ethylene butyrate, polyanhydrides, polydaricol acid, collagen, polyorthoesters, and polylactic acid. Such materials are available from ALZA Corporation (Mountain View, CA) and NOVA Pharmaceuticals, Inc. (Lake Elsinore, CA), and can also be readily prepared by those skilled in the art.
  • Ribosome suspensions can also be used as pharmaceutically acceptable carriers.
  • Useful ribosomes are prepared as a lipid composition containing, but not limited to, phosphatidylcholine, cholesterol and PEG-derived phosphatidylethanol (PEG-PE) through a filter of appropriate pore size so that it is sized for use, Purified by reverse phase evaporation.
  • PEG-PE PEG-derived phosphatidylethanol
  • Fab ′ fragments of antibodies can be bound to ribosomes via a disulfide exchange reaction (Martin and Papahadjopoulos, 1982).
  • the appropriate dosage level depends on the condition of the patient to be administered, the administration method, etc. If so, it can be easily optimized. In the case of injection administration, for example, it is generally preferred to administer from about 0.1 ⁇ g Zkg to about 500 mg Zkg per day of the patient's body weight, and would generally be administered in one or more divided doses. Preferably, the dosage level is from about 0.1 ⁇ g / kg to about 250 mgZkg per day, more preferably from about 0.5 to about 10 mg / kg.
  • the composition is preferably provided in the form of a tablet IJ containing preferably 1.0 to 10 mg of the active ingredient, and preferably has a productive force of .0, 5. 0, 10. 0, 15 0, 20. 0, 25. 0, 50. 0, 75. 0, 100. 0, 150. 0, 200. 0, 250. 0, 300. 0, 400. 0, 500. 0, 600. 0 , 750.0, 800.0, 900.0 and 1000.
  • the compound is administered on a regimen of! To 4 times a day, preferably once or twice a day.
  • a pharmaceutical composition or formulation should consist of uniform unit doses that ensure a certain dose.
  • a unit dose is a unit formulated with a pharmaceutically acceptable carrier, including a single dose effective for treating a patient.
  • the unit dosage of the present invention it is influenced by the physical and chemical characteristics of the compound to be formulated, the expected therapeutic effect, and the formulation considerations specific to the compound. Receive.
  • the method for introducing a nucleic acid molecule disclosed in the present invention for example, a cgrl polynucleotide, a vector having the polynucleotide inserted therein, and an antisense nucleic acid against the cgrl polynucleotide
  • a nucleic acid molecule disclosed in the present invention for example, a cgrl polynucleotide, a vector having the polynucleotide inserted therein, and an antisense nucleic acid against the cgrl polynucleotide
  • a nucleic acid molecule disclosed in the present invention for example, a cgrl polynucleotide, a vector having the polynucleotide inserted therein, and an antisense nucleic acid against the cgrl polynucleotide
  • in vivo delivery it is injected directly into the site of the patient in need of treatment.
  • ex vivo treatment cells at
  • the technique available for introducing a nucleic acid molecule into a living cell is selected depending on the force introduced in vitro into cultured cells or the like or whether it is introduced into a patient in vivo. Suitable techniques for introducing nucleic acid molecules into mammalian cells in vitro include ribosomes, electopore position, microinjection, transfusion, cell fusion, the DEAE-dextran method, and the calcium phosphate precipitation method. Transfection involves the binding of a recombinant viral (preferably retroviral) particle to a cellular receptor, followed by introduction of the nucleic acid molecule contained in the particle into the cell.
  • a commonly used vector for ex vivo delivery of genes is a retrovirus.
  • nucleic acid transfer techniques are viral or non-viral vectors (adenoviruses, lentiviruses, herpes simplex virus I, or adeno-associated viruses (AAV)), and cationic lipid-based Lipids (useful for lipid-mediated transfer of genes are eg D0TMA, DOPE, and DC_Chol; see, for example, Tonkinson et al., Cancer Investigation, 14 (1): 54_65 (1996))
  • the system is included.
  • the most preferred vector for use in gene therapy is a virus, among which is most preferably an adenovirus, AAV, lentivirus or retrovirus.
  • Viral vectors include at least one transcription promoter / enhancer or positioning factor.
  • a viral vector such as a retroviral vector, for example, a nucleic acid that functions as a cis element, that is, a translation initiation sequence, enables translation of the encoded gene when it is transcribed in a state containing a narcolepsy-related gene.
  • a viral vector such as a retroviral vector, for example, a nucleic acid that functions as a cis element, that is, a translation initiation sequence, enables translation of the encoded gene when it is transcribed in a state containing a narcolepsy-related gene.
  • Such vector constructs contain a packaging signal, a terminal repeat (LTR) or part thereof suitable for the virus used.
  • these vectors usually contain a signal sequence that causes the expressed polypeptide to be secreted from the host cell containing the vector.
  • the signal sequence for this purpose is a mammalian signal
  • the vector construct also includes a polyadenylation as well as a translation termination marker IJ.
  • a polyadenylation as well as a translation termination marker IJ.
  • it includes a 5 ′ LTR, a tRNA binding site, a packaging signal, an initiation point for DNA synthesis, and a 3 ′ LTR or a portion thereof.
  • Other non-viral vectors can use, for example, cationic lipids, polylysine, and dendrimers.
  • nucleic acid used for treatment may be provided with a reagent that targets the cell of interest, such as an antibody specific for a cell surface membrane protein or a receptor ligand on the target cell.
  • a reagent that targets the cell of interest such as an antibody specific for a cell surface membrane protein or a receptor ligand on the target cell.
  • the pharmaceutical composition can be included in the kit, container, pack along with instructions for administration.
  • the pharmaceutical composition according to the present invention is supplied as a kit, different components of the pharmaceutical composition are packaged in separate containers and mixed immediately before use. In this way the components They are packaged separately to allow long-term storage without losing the function of the active component.
  • Reagents contained in the kit are supplied in any type of container in which the components remain active for an extended period of time, are not adsorbed by the container material, and are not subject to alteration.
  • a sealed glass ampoule contains a buffer packaged under a neutral, non-reactive gas such as nitrogen gas.
  • Ampoules are composed of organic polymers such as glass, polycarbonate, polystyrene, ceramics, metals, or any other suitable material commonly used to hold reagents.
  • Examples of other suitable containers include simple bottles made from similar materials such as ampoules, and packaging materials that are internally lined with foil such as aluminum or alloys.
  • Other containers include test tubes, nozzles, flasks, bottles, syringes, or the like.
  • the container has a sterile access port such as a bot- nore with a stopper that can be penetrated by a hypodermic needle.
  • the kit also includes instructions for use. Instructions for using the kit comprising the pharmaceutical composition are printed on paper or other material and / or floppy disk, CD-ROM, DVD-ROM, Zip disk, video tape, audio tape, etc. It may be supplied as an electrically or electromagnetically readable medium. Detailed instructions for use may actually be included in the kit or may be posted on a website designated by the kit manufacturer or distributor or notified by e-mail, etc. .
  • the cgrl l gene (including not only the etason region but also the intron region and the promoter region) or cgrl l polypeptide variation (eg, point mutation, deletion, etc.), mutation in the expression control region of the gene
  • cgrl l gene including not only the etason region but also the intron region and the promoter region
  • cgrl l polypeptide variation eg, point mutation, deletion, etc.
  • a test sample is obtained from a patient suspected of having an abnormality in the proliferation and survival of specific cells, the antibody is brought into contact with the test sample, and the test sample and the antibody By detecting the presence or absence of binding, it is possible to examine the onset or possibility of onset of abnormalities in the proliferation and survival of the cells.
  • the presence or absence of binding between the test sample and the antibody can be confirmed by immunoprecipitation using antibodies, Western blotting, immunohistochemistry, ELI SA, and the like.
  • the polypeptide of the present invention is not detected at all or it is determined that the polypeptide is present in an extremely small amount in the living body, the onset of the abnormality is suspected.
  • primers, probes, etc. that can anneal with the cDNA sequence
  • genomic DNA sequence including transcriptional control region, intron region, etc.
  • Primers that can be used are generally 15 bp to: OO bp, and preferably ⁇ has a length of 17 bp to 30 bp.
  • the probe that can be used generally has a length of 15 bp or more, and hybridizes with at least a partial region of the coding region and non-coding region (including transcription control region, intron region, etc.) of the gene of the present invention. Anything that can be used can be used. Further, in order to confirm that the probe hybridizes to the target DNA region, the probe can be used in a state where it can be detected by a fluorescent dye, a radiolabel or the like.
  • Other methods for confirming functional abnormality of the cgrl 1 gene include, for example, obtaining a test sample from a subject and preparing the mRNA of the cgrl 1 gene prepared from the test sample, or preparing the mRNA force thereof.
  • a step of amplifying the partial region is included.
  • abnormalities were found in the expression level of the gene of the present invention compared to samples derived from healthy subjects. If possible, the onset or risk of dysfunction of the cgrl l gene can be predicted.
  • “mammal” means any animal classified as a mammal, and is not particularly limited. For example, in addition to humans, pet animals such as dogs and cats, ushi, pigs, hidges, horses, etc. It is a domestic animal. Particularly preferred “mammals” are humans.
  • the present invention includes a method for preventing or treating a diseased mammal suffering from or at risk of suffering from the disease.
  • treatment means to prevent or alleviate the progression of the pathological condition of a disease in a mammal that is likely to suffer from or suffers from the disease. It is used as a broad meaning including.
  • disease includes all pathological conditions caused by the presence or quantitative fluctuation of cgrl l gene or polypeptide, such as blood flow disorder, developmental disorder-related disease, neurological disease, cancer, hypotension. , Hypertension, and heart disease.
  • “mammal” means any animal classified as a mammal, and is not particularly limited.
  • pet animals such as dogs and cats, ushi, pigs, hidges, horses, etc. It is a domestic animal.
  • Particularly preferred “mammals” are humans.
  • the gene encoding the protein corresponding to spot2 in Fig. 1 is cgr 11 and has an EF-hand motif, and it has been reported that p53 suppresses cell proliferation.
  • this protein (cglrl polypeptide) has a secretion signal and is cleaved at the position of the secretion signal, and the measured molecular weight and the estimated molecular force of the protein are larger than expected. Therefore, it was guessed that it was modified with sugar. This indicates that this protein is a new secreted protein compared to previous reports.
  • Figure 3 shows the alignment of the amino acid sequences of rat and human derived cglrl polypeptides.
  • rat cgrl l was forcibly expressed in the cells (using the construct shown in FIG. 10), and the N-terminal amino acid sequence of the cgrl l protein purified from the culture supernatant was analyzed according to a conventional method. It was revealed that the signal peptide region is composed of 29 amino acid sequences (Fig. 5). This finding is different from the conventionally known cleavage rules for signal peptide sequences. Therefore, this result reveals the in vivo functional region of cgrl l protein for the first time and is an important finding.
  • HRE hypoxia response element
  • An antibody was prepared by synthesizing a peptide against the hydrophilic site of the amino acid sequence deduced from the gene sequence.
  • the site (sequence site shown as an antigenic site) present in FIGS. 3 and 5 was effective as an antigen, and a specific antibody was successfully produced. This part can be applied to measure the abundance of cgr 11 in the future.
  • PCR showed that cgrl 1 was specifically expressed in pituitary hormone-producing cells (MtTZS) (Fig. 7). This suggests that cgrl l protein is strongly related to the secretory function of cells. It was also suggested that it can be used to measure the secretory ability of hormones in vivo by using the abundance of cgrl l protein as an index.
  • cgrl l protein was specifically present in the Golgi apparatus (Fig. 8), supporting the above view that cgrl l polynucleotide was a secreted protein. Furthermore, the distribution was consistent with the distribution of growth hormone in hormone-producing cells (Fig. 8). Immunocytochemistry using an antibody specific for cgrl 1 protein revealed that cells with cgrl 1 protein were the anterior lobe after the pituitary gland and were not expressed at all in the pituitary midlobe ( Figure 9A). ).
  • cgrl l protein is abundantly expressed in sites with abundant blood vessels (posterior pituitary, anterior lobe), while it is not expressed at all in the region without any blood vessels (pituitary midlobe). It was strongly suggested that there was not.
  • the expression of cgrl 1 protein was also confirmed in the large follicle (Fig. 9C) and the outer follicular membrane of mature ovarian follicles (Fig. 9D). All of these sites are rich in blood vessels, suggesting that the cgrl l protein plays an important role in the formation and / or maintenance of the vasculature.
  • FIG. 10 shows the expression vector construct.
  • the cgrl 1 gene was inserted into the EcoRI and Xbal sites of the vector pCl_neo Vector (Promega) used for expression, and subcloning was performed according to a conventional method.
  • the resulting expression vector uses Lipofectamine 2000 It was introduced into pituitary-derived cell lines, MtT / E—B3 cells (MtT / E cells registered in RIKEN BioResource Center: subclone cells of RCB1278) and MtT / Se cells (registered RIKEN Bioresource Center: RCB0529).
  • cgrl l forced expression cells the cgrl l protein was localized in the gonoredi apparatus (FIGS. 11A and C), suggesting that the cgrl l protein also functions as a secreted protein in the forced expression cells. Furthermore, the presence of cgrl l protein was confirmed in the culture medium, strongly suggesting that cgrl l protein is a secreted protein (Figs. 12 and 13).
  • cgrl 1 gene product was forcibly expressed in MtTZE- B3 cells, and the obtained cells were transplanted subcutaneously into the groin of rats (Fischer344: female, lOOg) (2xl0 6 cells) Z200 ⁇ 1 / rat). On the 20th day after transplantation, the animals were laparotomized, the tumor was removed, and its weight was measured.
  • PC12 cells which are well known as neural progenitor cells in adrenal pheochromocytoma
  • PC12 cells are known to be unable to survive in serum-free media. Since the expression of cgrl 1 protein is low in this cell, it was introduced into PC12 cells into which the cgrl 1 gene was introduced, and a forced expression system was constructed. Inoculate 48 well plates with a forced expression clone (Fig. 17; clones C to G) and a non-forced clone (Fig. 17; control) with 5.00E + 4Z (200 ⁇ ), 2 The serum-free medium was replaced after a day. The number of viable cells was counted on day 8 of serum-free culture. As a result, it was revealed that the survival of PC12 cells in which cgrl l protein was forcibly expressed was significantly promoted even in serum-free medium (Fig. 17).
  • cgrl l protein is highly expressed in sites with blood vessels rich in the posterior and anterior pituitary glands, while it is not expressed at all in sites without blood vessels (pituitary middle lobe) and the colon (Fig. 9c) and the outer follicular membrane of mature ovarian follicles (Fig. 9D) have also been confirmed to express cgrl 1 protein, and cgrl l protein plays an important role in the formation and / or maintenance of vasculature. It has been suggested.
  • the left thigh was incised and the left femoral artery was detached. Hemostasis was achieved by fixing the left femoral artery with tweezers. A suture was passed through the proximal and distal sides of the left femoral artery. A hole was made in the femoral artery with a Weeckenor's scissors, and the PE10 side of a blood pressure measuring force neule filled with 500 U of Parin (Mochida Pharmaceutical) Z saline was inserted. The force needle and the blood vessel were fixed with the suture thread that had been passed through earlier, and it was confirmed that blood entered the tube while pulsating.
  • Parin Parin
  • the force neulet was delivered to the back of the head through the subcutaneous site. At this time, I was careful not to break the power. Once again, it was confirmed that blood was rising while pulsating, and stoppered with a stopper (length 2 cm, diameter 0.66 mm) (Silver Scale No. 16, TRAY). Subcutaneous chew The tube that touched the skin on the back of the head was squeezed with gum tape to fix the hub, the gum tape and the skin were sewn, and the tube exiting from the back of the head was cut to about 4 cm. One day after the operation, the blood pressure was measured by the following method under non-anesthesia after the animal had recovered.
  • the rat was placed in a cage, a blood pressure measuring force neuron and an extension tube were connected, and connected to a transducer (Bioresearch Corporation).
  • the tube was filled with 10 U of Parin Z physiological saline, and care was taken to prevent air from entering.
  • Stainless steel pipes were used for the connection between the force neulet and the extension tube.
  • PowerLab Bioresearch Corporation was used as blood pressure measurement and analysis software.
  • the cgrl1 protein (0.1 ⁇ g / 500 ⁇ 1) purified with an antibody from the culture medium of cgrl1 protein-secreting cells was injected into the abdominal cavity of the rat, and the time course of mean blood pressure was measured. As a result, an increase in blood pressure was observed after the purified cgrl l protein was injected intraperitoneally (Fig. 20). On the other hand, no increase in blood pressure was observed when a control sample was injected using a culture solution containing no purified cgrl protein.

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Abstract

L’invention porte sur une composition ou une méthode de favorisation de la croissance cellulaire, de soutien vital ou d’inhibition de la croissance d’une tumeur. On a découvert une nouvelle activité de la protéine cgr11 relativement différente des activités physiologiques de cette protéine rapportées jusqu’à présent. L’invention concerne une composition qui peut favoriser la croissance cellulaire et peut être impliquée dans le soutien vital, basée sur cette découverte. La composition comprend un polypeptide cgr11, un nucléotide qui encode le polypeptide, un antagoniste du polypeptide ou similaire. L'invention concerne également une méthode pour le traitement, la prévention ou le diagnostic d’une maladie.
PCT/JP2006/304969 2005-03-15 2006-03-14 Nouvelle proteine secretoire derivee d’une cellule pituitaire et utilisation de ladite proteine Ceased WO2006098304A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108220443A (zh) * 2018-01-31 2018-06-29 北京泱深生物信息技术有限公司 Cgref1作为标志物在肾透明细胞癌诊疗中的应用

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2000512494A (ja) * 1996-05-29 2000-09-26 ジェンザイム・コーポレーション 細胞増殖調節遺伝子

Patent Citations (1)

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Publication number Priority date Publication date Assignee Title
JP2000512494A (ja) * 1996-05-29 2000-09-26 ジェンザイム・コーポレーション 細胞増殖調節遺伝子

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Title
MADDEN S.L. ET AL.: "Induction of cell growth regulating genes by P53", CANCER RES., vol. 56, no. 23, 1996, pages 5384 - 5390, XP002046025 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108220443A (zh) * 2018-01-31 2018-06-29 北京泱深生物信息技术有限公司 Cgref1作为标志物在肾透明细胞癌诊疗中的应用

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