WO2006016571A1

WO2006016571A1 - Remedy for endometriosis

Info

Publication number: WO2006016571A1
Application number: PCT/JP2005/014559
Authority: WO
Inventors: Yutaka Osuga; Yasushi Hirota; Sadatoshi Sakuma
Original assignee: Cell Signals Inc.
Priority date: 2004-08-13
Filing date: 2005-08-09
Publication date: 2006-02-16
Also published as: JP2007297282A

Abstract

By studying the mitogen activity of MK on cultured endometrial stromal cells, it is clarified that MK stimulates the proliferation of ESC. By evaluating the MK concentration in the peritoneal fluid (PF), it is clarified that the MK level is elevated in a female with the progress of endometriosis. It is further found out that the MK concentration in the PF of a female under the therapy with GnRH agonist is significantly lower than the concentration in another group. It is indicated that MK mRNA is expressed in cells originating in peritoneal bone marrow, the peritoneal membrane and an intrauterine tissue.

Description

Treatment for endometriosis

Technical field

TECHNICAL FIELD [0001] The present invention relates to a drug for preventing or treating endometriosis containing a compound that suppresses mitdocaine as an active ingredient, and a test method and a test drug for endometriosis using the expression of mitdocaine as an index.

Background art

[0002] Endometriosis is a mysterious disease that impairs the health of women of reproductive age (Mo moeda et al., 2002; Osuga et al., 2002). It is widely believed that the cause of this disease is the colonization, survival and proliferation of the eclamptic endometrial tissue in retrograde menstruation in the abdominal cavity. However, this hypothesis cannot answer the question of why only a small percentage of women have endometriosis, despite the fact that most women have retrograde menstruation (Halme et al., 1984). ). Among the various abnormalities observed in women with endometriosis, much evidence suggests that changes in the abdominal environment are crucial for the onset of the disease (Harada et al., 200 l; Lebovic Et al., 2001). In addition, the concentrations of growth factors such as TNF, HGF, SCF, and IFN-γ, or cytodynamic force-in, change in the peritoneal fluid of endometriosis women (hereinafter referred to as PF) compared to healthy women. (Koga et al., 2000; Osuga et al., 1999; Osuga et al., 2000; Yoshino et al., 2003) o These substances are involved in cell survival, cell proliferation, and angiogenesis! It has been suggested to have various roles and stimulate the progression of endometriosis.

[0003] Mitodocaine (hereinafter referred to as MK) is a molecule having pleiotropic activities such as cell proliferation, cell migration, angiogenesis, induction of inflammation and fibrinolysis (Muramatsu, 2002; Horiba et al., 2000). ; Takada et al., 1997). Functions such as promoting cell proliferation, angiogenesis and inflammation have all been suggested to be important in the development of tumor-like lesions such as tumors and endometriosis. In fact, high levels of MK have been reported in many malignancies and have been suggested to be associated with the development of cancer (Garver, Jr. et al. 1994; Tsutsui et al., 1993; Nakagawa et al., 1995; O ' Brien et al., 1996; Koide et al., 1999; Aridome et al., 1995; Kato et al., 2000; Konishi et al., 1999). MK is also detected in eclampsia and its levels are comparable to healthy women. Compared with orthotopic endometrium, ectopic endometrium has a significantly lower level of MK compared to orthotopic endometrium. It is known that when it is expressed! / (See Non-Patent Document 1).

Non-Patent Document 1: Chung HW et al, Mol Hum Reprod 8, 350-355 (2002)

Disclosure of the invention

Problems to be solved by the invention

[0004] The present invention has been made in view of such a situation, and the purpose thereof is to clarify that MK is involved in the development of eclampsia and to prevent eclampsia that targets MK. It is also intended to provide a drug for treatment and a test method and a test drug for endometriosis.

Means for solving the problem

[0005] The present inventors have intensively studied to solve the above problems. First, the present inventors consider that cultured endometrial stromal cells (hereinafter referred to as ESC) are thought to be stimulated not only by MK derived from eclamptic endometrial cells but also by MKs of other origins. It was revealed that MK stimulates ESC proliferation by examining the mitogenic activity of MK. Next, by evaluating the MK concentration in PF, we revealed that MK levels increased in PF in women with advanced endometriosis. In addition, after treatment with GnRH agonists, it was found that MK concentrations in female PF were significantly lower than those in other groups! It was also found that MK mRNA is expressed in peritoneal bone marrow derived cells (hereinafter referred to as PBMC), peritoneum and endometrial thread and tissue.

These findings suggest that MK is involved in the onset and progression of eclampsia. Therefore, by inhibiting MK, endometriosis can be prevented or treated, and endometriosis can be examined using MK expression as an indicator.

That is, the present invention provides an invention including the following [1] to [4].

[1] A drug for preventing or treating endometriosis, comprising as an active ingredient a compound that suppresses expression of the mitdocaine gene.

[2] including a step of measuring the expression of the Midokine gene in a sample collected from the subject, Endometriosis testing method.

[3] A reagent for endometriosis examination comprising an anti-mitodocaine antibody.

[4] A reagent for endometriosis testing, comprising an oligonucleotide that hybridizes to the mitodocaine gene region and has a chain length of at least 15 nucleotides.

Brief Description of Drawings

[0007] Fig. 1 shows MK-induced proliferation of cultured ESC. The effect of MK on ESC proliferation was examined by measuring BrdU incorporation into DNA by using cell proliferation ELISA. ESCs were treated with different concentrations of MK for 24 hours. The value is 5 averaged SEMs per sample. Data shown are representative of 3 experiments using different ESC preparations. *; p <0.05, **; p <0.001 (both relative to control).

[Figure 2] Healthy women, women with mild eclampsia (r-ASRM score, 0-5, n = 58), women with progressive eclampsia ( _r -ASRM score, ≥6, n = 69), and MK concentration in PF of women treated with GnRH agonist (GnRHa, n = 12). The square frame represents the distance from the first quartile (25%) to the third quartile (75%), and the horizontal line in the square represents the median. The bar represents the 10th percentile at the lower limit and the 90th percentile at the upper limit.

FIG. 3 is a photograph showing the expression of MK mRNA in PBMC, peritoneum and eclampsia intimal tissue. Total RNA isolated from bone marrow-derived cells, peritoneum, and endometrial tissue was reverse transcribed and amplified by PCR using MK primers. GAPDH amplification was used to confirm RNA quality and quantity. Lanes 1-3: Bone marrow-derived cells in PF. Lanes 4-6: Abdominal tissue. Lanes 7-9: endometrial tissue. Lane 10: negative control without cDNA. Lane 11: DNA molecular weight Ma ¹ ~~ force` ~~.

BEST MODE FOR CARRYING OUT THE INVENTION

[0008] The present invention provides a drug for preventing or treating endometriosis comprising a compound that suppresses the expression of the MK gene as an active ingredient.

MK gene as a factor associated with endometriosis preferably has the ability to mention the human MK gene shown in SEQ ID NO: 1. It is not limited to this. 'Similar DNA encoding inducible protein is also known as' endometriosis Included in the MK gene as a “joint factor”. The sequence functionally equivalent to the DNA described in SEQ ID NO: 1 includes mutants derived from the same species, MK gene homologues derived from different species, and the like. For example, the previously reported mouse MK gene (Kadomatu, K., et al., Biochem. Biophy. Res. Commun., 151, 1312 (1988)) is functionally related to the DNA described in SEQ ID NO: 1. It is an example of an equivalent sequence, and is included in the MK gene as an eclampsia-related factor of the present invention.

[0009] Such a sequence functionally equivalent to the DNA described in SEQ ID NO: 1 is, for example, SEQ ID NO:

: A DNA that hybridizes with a DNA comprising the nucleotide sequence of 1 under stringent conditions and encodes a polypeptide having an activity of inducing eclampsia. Such DNA includes a degenerate mutant of the DNA represented by SEQ ID NO: 1.

[0010] The human-derived MK gene described in SEQ ID NO: 1 is prepared from a human cDNA library with a sequence having high homology with the sequence described in SEQ ID NO: 1 or a part thereof. This can be carried out by selecting a target and recovering from the selected clone. As the human cDNA library for obtaining the MK gene, for example, those constructed based on human kidney, human fetal kidney, brain, connective tissue, or total RNA derived from these cultured cells can be used. In addition to being obtained from nature, it may be synthesized by a DNA synthesizer based on the sequence described in SEQ ID NO: 1.

[0011] In addition, the preparation of a sequence functionally equivalent to the DNA of SEQ ID NO: 1 is performed by cDNA live derived from non-human mammalian cells (eg, human, monkey, mouse, rat, pig, ushi). From a rally, it can be carried out by selecting clones that can be hybridized under stringent conditions with the sequence of SEQ ID NO: 1 or a part thereof. Those skilled in the art can appropriately select the conditions for hybridization. For example, the conditions for hybridization are preferably highly stringent conditions. High stringency conditions are, for example, 65 ° C, 0.133 and 0.1% 303. Under these conditions, it is expected that DNA having high homology can be efficiently obtained as the temperature is increased. However, there are a number of factors that influence the stringency of the nodular and hybridization, such as temperature and salt concentration, and those skilled in the art can select similar factors by selecting these factors as appropriate. It is possible to realize genies.

In addition, the preparation of a sequence functionally equivalent to the DNA described in SEQ ID NO: 1 should be performed as described above. In addition to the method of selecting from the existing ones, the DNA having the sequence ability described in SEQ ID NO: 1 can be appropriately modified by genetic engineering and prepared.

[0012] The MK polypeptide as an eclampsia-related factor is preferably a force capable of mentioning the MK polypeptide encoded by the DNA described in SEQ ID NO: 1. SEQ ID NO: not limited thereto : A polypeptide having an endometriosis-inducing activity comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted or added in the amino acid sequence of the MK polypeptide encoded by the DNA of 1. Can be included.

[0013] A polypeptide functionally equivalent to the above MK usually has high homology in amino acid sequence with MK. High homology usually means at least 50% identity, preferably 75% identity, more preferably 85% identity, more preferably 95% identity at the amino acid level. Refers to sex. The identity of amino acid sequences and base sequences can be determined by BLAST (Proc. Natl. Acad. Sci. USA 90: 5873-5877, 1993).

[0014] One or more amino acids are mutated in the amino acid sequence of MK, and a polypeptide functionally equivalent to the polypeptide is also included in the present invention. The number of amino acids to be mutated is usually within 30 amino acids, preferably within 15 amino acids, more preferably within 5 amino acids (eg, within 3 amino acids), and even more preferably within 2 amino acids. The These mutated amino acid residues are preferably mutated to another amino acid that preserves the properties of the amino acid side chain. For example, amino acid side chain properties include hydrophobic amino acids (A, I, L, M, F, P, W, Y, V), hydrophilic amino acids (R, D, N, C, E, Q, G , H, K, S, T), an amino acid having an aliphatic side chain (G, A, V, L, I, P), an amino acid having a hydroxyl group-containing side chain (S, T, Υ), a sulfur atom-containing side Amino acids with chains (C, M), amino acids with carboxylic acid and amide-containing side chains (D, N, E, Q), amino acids with base-containing side chains (R, K, Η), aromatic-containing side Amino acids having a chain (H, F, Y, W) can be mentioned (both in parentheses represent single letter amino acids).

[0015] One of the compounds that suppresses the expression of the MK gene in the present invention includes nucleotides that can suppress the expression of the MK gene.

The first aspect of the nucleotide capable of suppressing the expression of the MK gene is as follows. An antisense oligonucleotide.

Antisense oligonucleotides include, for example, antisense oligonucleotides that hybridize to any position in the base sequence of SEQ ID NO: 1. This antisense oligonucleotide is preferably an antisense oligonucleotide against at least 15 or more consecutive nucleotides in the base sequence of SEQ ID NO: 1. More preferably, it is an antisense oligonucleotide in which at least 15 or more consecutive nucleotides contain a translation initiation codon.

[0016] Antisense oligonucleotides include not only nucleotides corresponding to nucleotides constituting a predetermined region of target MK DNA or mRNA, but all nucleotides complementary to each other, and oligonucleotides and bases represented by SEQ ID NO: 1. As long as it can specifically hybridize to a sequence, one having one or more nucleotide mismatches is also included.

[0017] According to the molecular design of an antisense oligonucleotide capable of suppressing MK gene expression, for example, based on the primary sequence of MK mRNA, a secondary structure having the minimum energy is predicted by a computer program. . Based on this predicted secondary structure, a loop portion that does not form a base pair can be selected as an antisense oligonucleotide sequence candidate. For example, the secondary structure prediction of human MK mRNA (GenBank Accession No. M69148: Tsutsui J. et al. Biochem. Biophys. Res. Commun., 176: 792-797, 1991) is performed by computer analysis. And (based on nucleotide numbers 15 to 32 of human MK mRNA sequence) (SEQ ID NO: 2), and an antisense DNA corresponding to a sequence that does not form a loop near the 5 'end (human MK mRNA sequence (SEQ ID NO: 3) can be designed.

SEQ ID NO: 2 5 '-AGGGTGAGGAGGAGGAAG-3'

SEQ ID NO: 3 5′—GAAGCCTCGGTGCTGCAT-3,

[0018] However, the oligonucleotide of the present invention can be appropriately designed based on the sequence of SEQ ID NO: 1, which is not limited to the sequence specifically shown above. Determination of whether or not the designed antisense oligonucleotide sequence has the activity of suppressing MK expression can be performed as follows. A substance with an antisense oligonucleotide sequence is administered to cells that synthesize and secrete MK, and MK is secreted into the culture medium by immunobiochemistry. Quantify to. Immunoassays for MK include enzyme immunoassay (Muramatsu, H. et al .: J. Biochem., 119: 1171-1175, 1996) or Western blotting after SDS polyacrylamide gel electrophoresis (Muramatsu, H. et al .: Dev. Biol, 159; 392-402, 1993) and quantification by densitometer. An antisense oligonucleotide that can reduce the production of the MK gene product by this quantification can be selected as one that can suppress the MK gene expression.

[0019] The term "oligonucleotide" t is also used to refer to naturally occurring oligomeric nucleic acid portions such as DNA and RNA deoxyribonucleotide and ribonucleotide structures, and to naturally occurring nucleic acids. Includes both artificial analogs capable of binding. Examples of the artificial analog include a lower alkyl phosphonate modification, a phosphorothioate modification or a phosphoramidate modification, a morpholino modification, a reverse-oriented thymidine modification, such as a methylphosphonate type or an ethylphosphonate type. Therefore, the oligonucleotide designed as described above may be modified by any of these as required. Such modified products have already been developed, and the already developed modified products may be applied to the therapeutic agent for eclampsia of the present invention. For example, morpholino modified products are disclosed in Japanese Patent Application Laid-Open No. 2003-210170, phosphorothioate modified products are disclosed in Japanese Patent Application Laid-Open No. 2002-142778, and reverse thymidine modified products are disclosed in Japanese Patent Application Laid-Open No. 2003-210171.

[0020] Of these modifications, phosphodiester-linked oligonucleotides are particularly susceptible to nuclease action in serum or inside cells. Therefore, a preferred embodiment of the antisense oligonucleotide of the present invention is a phosphorothioate-linked or methylophosphonate-linked analog that is nuclease resistant (Stein et al., Cancer Research 48: 2659, 1998).

Such oligonucleotide artificial analogs are prepared by methods well known to those skilled in the art, for example, methods using commercially available machines and reagents available from Perkin-Elmer / Applieds Biosystem (Foster City, CA). be able to.

[0021] In another embodiment of the present invention, the antisense oligonucleotide may not be in the form of an oligonucleotide itself, for example, after the target cell is infected as an expression construct mounted on an expression vector or the like. It can be generated as an RNA molecule. The antisense oligonucleotide of the present invention is applied to a patient so that it can reach the affected area by force applied directly to the affected area of the patient or by administration into the blood vessel. Furthermore, an antisense encapsulating material that enhances durability and membrane permeability can also be used. Examples include liposomes, poly-L-lysine, lipids, cholesterol, lipofectin, or derivatives thereof.

The dosage of the antisense oligonucleotide of the present invention can be appropriately adjusted according to the patient's condition, and a preferred amount can be used. For example, it can be administered in the range of 0.1 to 100 mg / kg, preferably 0.1 to 50 mg / kg.

[0022] The second embodiment of the nucleotide that suppresses the expression of the MK gene includes dsRNA targeting the MK gene.

The “dsRNA targeting the MK gene” means a dsRNA that can suppress the expression of the MK gene by RNA interference (RNAi). One strand of dsRNA for suppressing MK gene expression with RNAi has a sequence complementary to at least MK mRNA. As long as it is a site where expression can be suppressed, it may have a complementary relationship in any region of MK mRNA. In addition, this complementarity may have a mismatch of about several bases with a target sequence that does not need to be complete as long as it can induce RNAi and suppress MK gene expression.

[0023] As the dsRNA, both forms of artificially synthesized dsRNA itself or dsRNA expressed in mammalian cells by an expression vector can be adopted.

When dsRNA is artificially synthesized and introduced into mammalian cells, it is necessary to consider the cytotoxicity of long dsRNA to mammalian cells, even if the dsRNA is expressed from an expression vector. . Therefore, both artificially synthesized dsRNA (hereinafter referred to as “synthetic dsRNA”) and dsRNA generated by expression should have a chain length of, for example, 19 to 30 base pairs, but this is longer than this. Even dsRNA can be used if it does not add cytotoxicity.

The above dsRNA targeting MK mRNA is paired with MK mRNA in vivo, and induces cleavage of MK mRNA by an in vivo enzyme. Therefore, translation of MK from mRNA to protein is inhibited, and as a result, expression of MK can be suppressed and the onset of endometriosis can be suppressed. [0024] As a second aspect of the present invention, there is provided a drug for preventing or treating endometriosis, which comprises a compound that suppresses the function of MK protein as an active ingredient.

The compound that suppresses the function of MK includes an antibody that binds to MK. An already developed anti-MK antibody (Muramatsu H. et al., Dev. Biol. 159: 392-402, 1993) that can suppress the action of MK is applied to the treatment and prevention of eclampsia in the present invention. May be. A monoclonal antibody against human MK has also been developed (Japanese Patent Laid-Open No. 2002-085058), and this may be applied. However, the antibodies of the present invention are not limited to these already reported antibodies, and anti-MK polyclonal antibodies or monoclonal antibodies prepared by the anti-MK antibody preparation method described in the later-described test method can also be used. .

[0025] It is also effective to use a recombinant antibody or a modified antibody as the MK antibody used as a therapeutic agent. As a recombinant antibody, for example, as a monoclonal antibody, the antibody gene is cloned by high-pridoma force, incorporated into an appropriate vector, and introduced into a host.

Recombinant antibodies produced using genetic recombination techniques can be used (for example, Borrebaeck CAK & Larrick JW, THERAPEUTIC MONOCLONAL ANTI BODIES, Published in the United Kingdom MACMILLAN PUBLISHERS LTD, 1990). As the modified antibody, for example, a chimeric antibody or a humanized antibody can be used. A chimeric antibody can be obtained by linking DNA encoding an antibody V region other than a human antibody and DNA encoding a human antibody C region, incorporating this into an expression vector, introducing it into a host, and producing it. (EP 125023, PCT WO96 / 02576) _o

The antibody used in the present invention may be a modified antibody fragment as long as it binds to MK and inhibits the activity of MK. For example, as an antibody fragment, Fab, F (ab '), Fv or H chain

2

And single chain Fv (scFv) in which Fv of L chain is linked with an appropriate linker.

[0026] As a third aspect of the present invention, there is provided a method for examining endometriosis, which comprises the step of measuring the expression of the MK gene in a sample collected from a subject.

MK is thought to be involved in the development of endometriosis, as will be described in the examples below. Therefore, in order to investigate the cause of endometriosis in patients and perform appropriate treatment for patients, it is useful to measure the expression of the MK gene as a test for endometriosis. As a sample collected from a subject, for example, PF, PBMC, peritoneal tissue or eclampsia intimal tissue can be used.

[0027] As a first embodiment in the measurement of MK gene expression, the translation product MK protein is measured. First, a protein sample is prepared from the above-described subject sample. Next, the amount of MK protein is measured from the protein sample. Compare the measured protein amount to a control value based on the expression level of MK in healthy subjects. As a result of this comparison, if the subject's MK protein is significantly elevated compared to the control, it is determined that the subject may or has already developed endometriosis. The

The above MK protein was measured by SDS polyacrylamide electrophoresis and Western blotting, dot blotting, immunoprecipitation, enzyme-linked immunoassay (ELISA), and immunoassay using antibodies to MK protein. A fluorescence method can be exemplified.

[0028] As another method for measuring the expression of the MK gene, the transcription product MK mRNA is measured. First, a total RNA sample is prepared from the subject sample. Total RNA can be prepared by methods well known to those skilled in the art. For example, it can be carried out by using a total RNA preparation kid Isogen "(Nitsubon Gene Co., Ltd.). Then, the amount of MK mRNA contained in the total RNA sample is measured. The amount of RNA measured is used as a control. Compare.

[0029] In another embodiment, a cDNA sample is prepared from a subject sample. A cDNA sample can be prepared by synthesizing cDNA using reverse transcriptase using the above-mentioned total RNA as a saddle. Next, the amount of MK cDNA contained in the cDNA sample is measured. The amount of cDN A measured is compared to the control.

Examples of methods for measuring gene expression based on these mRNAs and cDNAs include methods well known to those skilled in the art, such as Northern blotting, RT-PCR, and DNA array.

If the mRNA or cDNA comparison results in a significant increase in the subject's MK protein mRNA or cDNA compared to the control, the subject may develop endometriosis, or It is determined that it has already developed.

[0030] As a fourth aspect of the present invention, a test reagent for use in a test method for endometriosis Is provided. Examples of such a test reagent include a test drug containing an oligonucleotide containing a partial sequence of the MK gene that can be used in the above test method (including a substrate on which an oligonucleotide probe is immobilized) Examples include test drugs containing MK antibodies. These antibodies and oligonucleotides may be labeled according to the test method.

[0031] As a first aspect of the test reagent for use in the endometriosis test method, a test reagent for eclampsia that contains an anti-MK antibody can be mentioned. The anti-MK antibody as the test agent may be, for example, a polyclonal antibody or a monoclonal antibody as long as it can detect MK. For example, in the case of a polyclonal antibody, an antibody already developed by the present inventors may be used (Muramatsu H. et al, Dev. Biol. 159: 392-402, 1993). Monoclonal antibodies have also been developed (Japanese Patent Application Laid-Open No. 2002-085 058) and can be suitably used.

[0032] Preparation of a specific antibody against MK can be performed according to a known method. Monoclonal antibodies can be obtained, for example, using the entire human MK polypeptide encoded by the DNA represented by SEQ ID NO: 1 as a sensitizing antigen, or a partial sequence having a length capable of retaining antigenicity as a sensitizing antigen. You can use it to make it. A hybridoma that produces a monoclonal antibody can basically be prepared as follows according to the method of Kohler and Milstein (Kohler G. & C. Milstein, Nature 256: 49 5-497, 1975). . That is, using the above-described MK protein or a part thereof as a sensitizing antigen, it is immunized according to a normal immunization method, and the resulting immune cell is fused with a known parent cell by a normal cell fusion method. It can be prepared by screening monoclonal antibody-producing cells by ordinary screening methods.

The mammal to be immunized with the sensitizing antigen is not particularly limited, but it is generally preferable to select the mammal in consideration of compatibility with the parent cell used for cell fusion. Animals such as mice, rats, hamsters and the like are used.

[0033] A second embodiment is a reagent for endometriosis testing that includes an oligonucleotide that hybridizes to the MK gene region and has a chain length of at least 15 nucleotides.

The oligonucleotide specifically hybridizes to DNA (normal DNA or mutant DNA) containing the MK gene region. Where “specifically hybridize” Are described under normal and abbreviated hybridization conditions, preferably under stringent hybridization conditions (e.g., described in Sambu Norec et al., Molecular Cloning. Cold Spring Harbor Laboratory Press, New York, USA, 2nd edition 1989). In the above condition) does not cause significant cross-hybridization with DNA encoding other proteins. The oligonucleotide need not be completely complementary to the DNA containing the MK gene region, if specific noidisation is possible! /.

[0034] Oligonucleotides that hybridize to DNA containing the MK gene region and have a chain length of at least 15 nucleotides can be used as probes (including the substrate on which the probe is immobilized) and primers in the test method of the present invention. it can. When the oligonucleotide is used as a primer, the length is usually 15 bp to 100 bp, preferably 17 bp to 30 bp. The primer is not particularly limited as long as it can amplify at least a part of the MK gene region.

[0035] When the oligonucleotide is used as a probe, the probe is not particularly limited as long as it specifically hybridizes to DNA containing the MK gene region. The probe may be a synthetic oligonucleotide and usually has a chain length of at least 15 bp.

The oligonucleotide of the present invention can be produced by, for example, a commercially available oligonucleotide synthesizer. The probe can be prepared as a double-stranded DNA fragment obtained by restriction enzyme treatment or the like.

[0036] When the oligonucleotide of the present invention is used as a probe, it is preferably used after being appropriately labeled. As a labeling method, T4 polynucleotide kinase is used to label the oligonucleotide by phosphorylating the 5 ′ end with ³² P, and a random hexamer oligonucleotide is used using a DNA polymerase such as a Talenow enzyme. Examples thereof include a method of incorporating a substrate base labeled with an isotope such as ³² P using a nucleotide or the like, a fluorescent dye, or piotin (such as a random prime method).

The ability to utilize the oligonucleotide of SEQ ID NO: 2 or SEQ ID NO: 3 as the oligonucleotide of the present invention The oligonucleotide of the present invention is not limited to the sequence specifically shown above. Number: Designed appropriately based on the sequence of 1 can do.

All prior art documents cited in the present specification are incorporated herein by reference.

Example

Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples.

1. ESC isolation, purification and culture

ESC primary cultures were prepared from biopsies by the method described in Ryan, IP et al. With some modifications. Endometrial tissue is separated from the underlying soft tissue force and subdivided into small pieces, and type I collagenase (Sigma, St. Louis, MO) and deoxyribonuclease I (Takara, Tokyo) in DMEM / F12 , Japan) at 37 ° C for 1-2 hours and separated using continuous filtration. Cell debris was removed with a 100 / zm Nylon cell strainer (Becton Dickinson, Lincoln Park, NJ) and some of the epithelial glands were removed with a 70 m nylon cell strainer (Beta Ton Dickinson). The stromal cells remaining in the filtrate are collected by centrifugation, resuspended in DMEM / F12 medium, seeded in a 100 mm dish, allowed to adhere for 30 minutes at 37 ° C, and then rinsed with phosphate buffered saline. As a result, non-adherent epithelial cells and blood cells were removed. Cells were cultured in DMEM / F12 medium supplemented with 10% activated carbon treated FBS (HyClone, Logan, Utah) and antibiotics (Sigma). After the cells reach confluence, dissociate the cells with 0.25% trypsin, collect them by centrifugation, seed them in a Falcon 96 multiwell plate (Bettaton Dickinson) at 1 x 10 ⁴ cells / well, and humidify. Maintained at 37 ° C in a 5% CO 2/95% air environment. 24hours

2

The stromal cell population was subsequently purified by immunocytochemical staining with the following antibodies (Dako); vimentin (stromal cells), cytokeratin (epithelial cells) and CD45 (single cells). Spheres and other white blood cells). The purity of the stromal cells was 98% or higher, judging from the positive cell staining for vimentin and the negative cell staining for cytokeratin and CD45.

[0038] 2. Collection of PF sample The subjects in this study included women (n = 106) and healthy women (n = 33) with eclampsia who were laparoscopic for pain and Z or infertility, aged 20-48 years. There are 13 9 people in total. Endometriosis was diagnosed by both laparoscope and histology. The degree of disease was scored according to the revised American Society for Reproductive Medicine (r-ASRM) classification system (American Society for Reproductive Medicine, 1997). In the non-endometriosis group, 14 women were in the follicular phase and 19 were in the luteal phase. The endometriosis group includes 12 women treated with GnRH agonist for more than 3 months before laparoscopy (GnRHa group). All women except the GnRHa group had a regular menstrual cycle; of these, 49 were in the follicular phase and 45 were in the luteal phase. In the group of eclampsia that has not received GnRHa treatment, the status of eclampsia is mild eclampsia (r-ASRM score, l-5; n = 25), and advanced eclampsia (r -ASRM score ≥6, n = 69), assuming that mild endometriosis is the onset stage of the disease. The age of women in the endometriosis group was 32.7 ± 4.1 years (mean SD), essentially the same as that of women in the non-endometriosis group (31.1 ± 5.6 years). This study was approved by the University of Tokyo Institutional Review Board, and consent was signed by each woman regarding sample collection.

[0039] PF also collected laparoscopic force-Eureka introduced into the Douglas fossa before any manipulation technique. PF was centrifuged at 400 xg for 10 minutes and the supernatant was stored frozen at -80 ° C until assembly. Peritoneal and endometrial tissues were also collected.

PBMC were collected by well-known methods (Yoshino et al., 2003). Briefly, the collected PF was centrifuged at 200 × g for 5 minutes to remove the supernatant. Cell pellets are resuspended in phosphate buffered saline (PBS) and overlaid on Ficoll-Pak (Amersh am Biosciences, Upsala, Sweden) at 150 X g for 30 minutes Centrifuged. PBMC were collected from the interface.

[0040] 3. Cell proliferation

Cell proliferation assay was performed as previously reported by the inventors (Tang et al., 2002). Effect on ESC proliferation by measuring 5-bromo-2'-deoxyuridine (BrdU) incorporation into DNA using Biotrack cell proliferation ELISA system (Amersham Biosciences) according to manufacturer's instructions The effect of MK was examined. In short, E SCs were seeded in Falcon 96 multiwell plates at a density of 1 × 10 ⁴ cells / well in 100 1 culture medium with different concentrations of MK (0, 10, 100 and 1000 ng / ml). After 24 hours, BrdU solution 1001 was added and incubated at 37 ° C for an additional 2 hours. After removing the culture medium, the cells were fixed, and DNA was denatured by adding a fixing solution 2001 / well. Peroxidase-labeled anti-BrdU antibody bound to BrdU incorporated into newly synthesized cellular DNA. Subsequent substrate reactions detected immune complexes and the resulting color was read at 450 nm in a digscan microplate reader (ASYS Neutech GmbH, Eugendorf, Austria).

[0041] 4. MK measurement

MK concentrations in PF were donated by Meiji Dairies (Odawara, Japan) or determined using the enzyme-linked immunoassay (EIA) for Cell Signals (Yokohama, Japan). The detection limit of this EIA was 0.1 ng / ml / sample.

[0042] 5. RNA extraction, MK mRNA reverse transcription-polymerase chain reaction (RT-PCR)

Total RNA was also extracted from PBMC, peritoneum, and endometrial tissue using the RN Easy Mini Kit (QIAGEN, Hilden, Germany). RT-PCR was performed using a lever 'tiger' dash (Toyobo, Tokyo, Japan). 1 g of total RNA was reverse transcribed in a total volume of 201 and cDNA was amplified using oligonucleotide primers based on the human MK sequence. Human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primer (Toyobo, Tokyo, Japan) was used to confirm RNA quality and quantity. To amplify the 320 bp fragment, an MK primer (sense, 5′-CCTGCAACTGGAAGAAGGAG-3 ′ (SEQ ID NO: 4); antisense, 5′-AGCAGACAGAAGGCACTGGT-3 ′ (SEQ ID NO: 5)) was selected. PCR conditions for amplification of MK and GA PDH were 10 seconds at 98 ° C, 2 seconds at 60 ° C and 30 seconds at 74 ° C for 20 seconds. PCR products were analyzed by agarose gel electrophoresis using ethidium bromide. Each PCR product was purified using the QIAEX II gel extraction kit (Qiagen) and the same using an ABI PRISM ™ 310 gene analyzer (Applied Biosystems, Foster City, Calif.). The sex was confirmed.

[0043] 6. Statistical analysis ANOVA was used to compare BrdU incorporation of cultured cells. MK data in the PF is described as the median and interquartile range (IQR). The MK concentration in PF was compared using the Mann-Whout-One test. A value of 0.05 is considered statistically significant.

[Example 1]

MK mitogenic activity on cultured ESCs was examined by measuring 5-bromo-2'-deoxyuridine (Br dU) incorporation. MK concentrations in PF in women with endometriosis or healthy women and in women treated with GnRH agonists were measured using the specific enzyme immunoassay. The expression of MK mRNA in PBMC, peritoneum and endometrial tissue was assessed by RT-PCR.

Figure 1 shows the effect of MK on DNA synthesis in ESC. BrdU incorporation into DNA was significantly increased by 100 and 1000 ng / ml MK. 1000 ng / ml MK enhanced BrdU incorporation level to 125% of control.

[0045] All PF samples examined contained detectable concentrations of MK above the Atsey limit.

MK concentrations in PF (0.93 ng / ml, 0.87 to 1.17; median, IQR) in women with mild eclampsia (stage I) were substantially the same as those in healthy women (0.97 ng / ml, 0.67-1.27). Therefore, we combined data from healthy women and women suffering from mild endometriosis to develop progressive epidural endometriosis (stage 11, III, and IV) (r-ASRM score ≥6) Compared to the data of other women. As shown in Figure 2, MK concentrations in PF (1.21 ng / ml, 0.80-2.27) in women with advanced endometriosis are MK concentrations in healthy women and women with mild endometriosis. (0.96 ng / ml, 0.67-1.26, p <0.05). MK concentrations in women receiving GnRHagost treatment were significantly lower than those in the other two groups (p 0.001). When MK concentrations in PF were compared according to menstrual period, the concentrations in the luteal phase (1.32 ng / ml, 0.72 to 2.21) were significantly higher than those in the follicular phase (0.95 ng / mU 0.68 to 1.24, p <0.05) .

As shown in FIG. 3, MK mRNA expression was detected as a clear band at 320 bp in PBMC, peritoneum, and eclampsia tissue.

[0046] In the present invention, the inventors have shown that MK stimulates ESC proliferation. In addition, MK levels in PF were increased in women with advanced endometriosis compared to women with mild eclampsia or healthy women. Interestingly, orthotopic endometrial cells in women with endometriosis express higher levels of MK compared to healthy women (Chung et al., 2002). Has also been demonstrated to express significantly lower levels of MK compared to the orthotopic endometrium (Chung et al., 2002). It may be useful to solve this dilemma if we consider promoting it. In particular, MKs derived from other sources other than ectopic endometrium may be involved in disease progression, but MK increased in orthotopic endometrium is very early stage of endometriosis May help in the establishment and survival of reflux endometrial cells.

[0047] A recent report shows that MK stimulates intraperitoneal adhesions in a murine abdominal injury model (Inoh et al., 2004). Given that they tend to form internal adhesions, MK may also be involved in adhesion formation.

The origin of MK in PF seems to be widely distributed. Our RT-PCR analysis demonstrated that MK is expressed in PBMC, peritoneum and endometrial tissue. Furthermore, the MK in the follicular fluid released during ovulation is assumed to constitute part of the MK in PF because the follicular fluid contains a large amount of MK (Ohyama et al., 1994). The high MK concentration in the follicular fluid! May partially explain the finding that the MK in PF is increased in the luteal phase compared to the follicular phase.

[0048] Notable findings in this study indicate that MK levels in women receiving GnRHa treatment are significantly higher compared to women with advanced endometriosis and mild endometriosis or healthy women That is low. Estradiol has been reported to induce MK mRNA expression in eclamptic intimal epithelial cells (Zhang et al., 1995). ₀ Several ERE half palindromes present in the promoter region of the MK gene (Uehara et al., 1992). It was suggested that the motif (Kato et al., 19 92) causes the action of estradiol (Zhang et al., 1995). The hypoestrogenic state induced by GnR Ha treatment may repress MK gene transcription in various cells. In addition, anovulation caused by GnRHa treatment may be involved in suppressing the influx of follicular fluid into the peritoneal cavity by suppressing MK levels in PF.

[0049] MK grows several cells, including fibroblasts, tumor cells, and keratinocytes (Inazumi et al., 1997; Muramatsu and Muramatsu, 1991; Mura matsu et al., 1993). However, the mechanism of action of MK is less clear because there is no clearly defined MK receptor capable of transmitting growth factor signals (Muramatsu, 2002). Thus, elucidating the mechanism by which MK stimulates eclampsia intimal cells appears to be difficult at this time. However, given that MK stimulates the onset of the disease, endometriosis may be treated with MK inhibitors. In mouse rectal cancer cells, antisense oligonucleotides against MK have been shown to suppress tumor formation (Takei et al., 2001).

Industrial applicability

In the present invention, it has been clarified for the first time that eclampsia endometrial cells are stimulated not only by MK derived from eclampsia endometrial cells but also by MKs from other sources. Furthermore, MK levels in PF have been shown to increase in women with advanced endometriosis compared to women with mild endometriosis or no endometriosis, and MK levels in PF Have been shown to be involved in the development and progression of eclampsia.

Based on the above findings, it is possible to provide a drug for preventing or treating endometriosis targeting MK, and an endometriosis test method and test drug using MK expression as an index.

Claims

The scope of the claims

[1] A drug for preventing or treating endometriosis, comprising as an active ingredient a compound that suppresses expression of a mitdocaine gene.

[2] The drug according to claim 1, wherein the compound that suppresses expression of the mitdocaine gene is an oligonucleotide.

[3] The drug according to claim 2, wherein the oligonucleotide capable of suppressing the expression of the mitodocaine gene is the following (a) or (b):

(a) An antisense oligonucleotide of the mitdocaine gene

(b) dsRNA targeting mitodocaine gene

[4] A drug for preventing or treating endometriosis, comprising as an active ingredient a compound that inhibits the function of mitodocaine protein.

[5] A compound that suppresses the function of mitdocaine protein is an antibody that binds to mitdocaine

The drug according to claim 4.

[6] A method for examining endometriosis, comprising a step of measuring the expression of a mitdocaine gene in a sample collected from a subject.

7. The examination method according to claim 6, wherein the sample collected from the subject is peritoneal fluid, peritoneal bone marrow derived cells, peritoneal tissue or endometrial tissue.

[8] The test method according to claim 6 or 7, wherein the measurement of mitdocaine gene expression is performed based on mitodocaine protein or mRNA encoding mitdocaine protein.

[9] A reagent for endometriosis testing comprising an anti-mitodocaine antibody.

[10] A reagent for endometriosis testing, comprising an oligonucleotide that hybridizes to a mitodocaine gene region and has a chain length of at least 15 nucleotides.