JP2002500888A - Expression and application of INSL4 gene in human embryonic bone tissue - Google Patents

Abstract

  (57) [Summary]   The present invention relates to a method for locating, detecting and diagnosing the expression of the insulin-like 4 (INSL4) gene in bone tissue and ligament of a human embryo, a method for labeling and detecting the tissue expressing an INSL4 gene expression product, and And a method for selecting a compound capable of regulating the activity of the compound. The invention also relates to diseases associated with the differentiation and / or abnormal proliferation of said tissue, in particular abnormal cartilage development and / or abnormal ossification of the bone during formation and / or abnormal development of the interarticular ligament. The present invention also relates to a medicine for treating an illness.

Description

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method for locating, detecting and diagnosing the expression of the insulin-like 4 (INSL4) gene in human embryonic bone tissue and ligaments, and to label and detect cells of the tissue expressing an INSL4 gene expression product. And a method for selecting a compound capable of altering the activity of the cell. The invention also relates to diseases which lead to abnormal growth and / or differentiation of said tissue, in particular to abnormal development of cartilage and / or abnormal ossification of bone during formation and / or abnormal development of inter-articular ligaments. And pharmaceutical products for treating

[0002] Insulin, IGF-I, IGF-II (insulin-like growth factors I and II) and relaxin share certain structures and functions in common, especially their effects on cell growth, development, differentiation and metabolism (D. Le Roith, 1997).

[0003] Insulin, as is well known, is a pancreatic endocrine hormone that regulates energy metabolism. Insulin-like growth factor IGF-I is a growth-promoting peptide expressed in many tissues and involved in endocrine, paracrine and autocrine regulation of cell growth. IGF-II has similar properties, but is more abundantly expressed during fetal life and is considered to be a fetal growth factor.

[0004] Relaxin induces connective tissue remodeling of the reproductive tract and suppresses uterine contractions.
The relaxin gene is expressed in the corpus luteum, decidua, trophoblast and prostate (Bogic,
LV et al., 1995). Very high expression has also been observed in the brain, but its functional role in the brain remains unclear.

[0005] Ley I-Ls (INSL3), which has now been cloned in cDNA form and has an unknown biological activity, has also been added to this family. Ley
IL transcripts are present in Leydig cells and other tissues such as the corpus luteum, trophoblast, fetal membrane and breast tissue (Adama, IM et al., 1993; Tashima, LS et
al., 1995).

[0006] The apparent importance of these genes in regulating fetal growth and stem cell development has been shown in various studies (VKM Han et al., 1996; DJ Hansell et al, 1991).
And LS Tashiman et al., 1995). IGF-I and IGF-II are expressed by most fetal and embryonic tissues and regulate cell growth and differentiation like autocrine / paracrine factors. Site-directed mutagenesis performed on genes encoding the growth factors IGF-I and IGF-II demonstrated considerable utilization of the growth factor system in the growth process (J. Baker et al., 1993; TM De Chiara et al., 1990 and JP Liu et al., 1993). De Chiara et al. Demonstrated a causal relationship between the knockout of the IGF-II gene and the growth-deficient phenotype that emerged from embryonic day 16 and persists after birth. . Liu et al is, (- I -) weight at birth of IGF-I mutant mice is about 60% of the normal body weight, some have found to be a stillborn.

This peptide family has common structural features defined by the positions of various cysteines that are essential for the formation of a tertiary structure. It has been possible to demonstrate that all members of this family bind to cell surface receptors. These receptors have been identified by molecular cloning and have been well characterized for insulin and IGFs. These receptors are receptors for growth factors and their cancerous analogs [e.g., c-erbB2 / neu (EGF
Receptor), c-met (hepatocyte growth factor receptor), fms (CSF-1 receptor) and trk (NGF
Receptors) and so on, and belongs to the superfamily of tyrosine kinase receptors (TKRs).

[0008] The intracellular signaling pathways for these receptors are characterized by tyrosine kinase activity that, following autophosphorylation of tyrosine residues of the receptor, results in a chain of events corresponding to phosphorylation. This includes in particular the activation of IRS-1 (especially in insulin and IGFs), P13K, Shc, GBR2, Sos, Ras, Raf and mitogen-activated protein kinase (MAP kinase), which phosphorylation It peaks when the cascade affects various cellular processes such as transcription.

[0009] The pleiotropic physiological effects of this signal cascade have generally been an intensive research topic. All polypeptides of this family member are signal peptides, B chains,
It is characterized by the presence of the connecting peptide C or C chain, as well as the A chain.

[0010] For these polypeptide hormones, the term preprohormone, prohormone or mature hormone is defined by the presence or absence of certain elements and their arrangement in the tertiary structure. Preprohormones differ from prohormones in that a signal peptide element is present. The difference between prohormones and active mature hormones is more complex and depends on the type of hormone considered.

[0011] For example, in proinsulin and prorelaxin, the B chain and the A chain are each N-chain.
Located at the termini and the C-terminus, which are separated by a long connecting peptide C, during the maturation phase the connecting peptide C is cleaved to the active form of the hormone.

[0012] Unlike insulin and relaxin, the C peptides of the IGFs are not cleaved during the maturation phase. The mature form of the IGF peptide comprises two domains in addition to the A, B, and C domains.
It contains two carboxy-terminal peptides (D and E domains), which are cleaved after expression.

[0013] Recently, a novel gene of the insulin-like family named INSL4 has been characterized (WO 95/34653; Chassin, D. et al., 1995). This gene is an early placenta cDNA
Cloned from the library, located on chromosome 9p24.

The INSL4 gene is known as “EPIL” (early placental insulin-like peptide, early placenta i
It encodes a 139 amino acid polypeptide designated nsulin-like peptide). For clarity, the term EPIL polypeptide is referred to as a putative 139 amino acid polypeptide derived from a coding nucleic acid sequence (as described in FIG. 1).
To mention.

[0015] The derived amino acid sequence is consistent with the general structure of this hormone family, including the signal peptide, B and A chains, and the connecting peptide C (or C chain).
And some structural homology, probably the presence of six cysteine residues that must be involved in the formation of the three disulfide bridges.

The above-mentioned document states that the constitution of the INSL4 gene consists of two exons and one intron. These references also show that mRNA transcripts of the INSL4 gene were detected only in placental tissues and weakly in the uterus (D. Chassin et al.
., 1995).

The INSL4 gene, which also belongs to this family, may be a novel member of this gene family involved in human development. Studies of the pathways of expression of these genes at various stages of development have led to a successful approach to better understand the role of these genes in embryonic and fetal development (T. Strachan et al., 1997;
C. Ottoleghi et al., 1997 and. Burn et al., 1995). INSL4 and INSL in the development of placenta and human embryonic tissue at the onset of pregnancy by in situ hybridization
In studies of the timing and location of expression of the IGF-II gene, the inventors have demonstrated high levels of expression of INSL4 transcripts in human trophoblasts, particularly in the syncytiotrophoblast layer.
Quite surprisingly, we also demonstrated higher levels of expression of INSL4 transcripts in 8-week-old fetal tissues, but only in the joint ligaments and perichondrium.

Accordingly, one subject of the present invention is characterized in that it is expressed in fetal bone tissue cells.
EPIL1, EPIL2 or EPIL3 polypeptide encoded by the INSL4 gene.

As used herein, the term “polypeptide” also refers to a protein or peptide. In the present invention, the term "EPIL1 polypeptide" includes the A, B and C regions of the entire amino acid sequence selected from the amino acid sequence from positions 18 to 139 (including both ends) of the amino acid sequence shown in FIG. It means a polypeptide encoded by the INSL4 gene constituting a single chain, and the entire sequence includes an amino acid sequence of at least positions 24 to 139 (including both ends). The EPIL1 polypeptide may also include a signal peptide, in which case the amino acid sequence of the EPIL1 polypeptide including the signal polypeptide may be from position 1 to position 139 (inclusive) of the amino acid sequence described in FIG. It is an amino acid sequence.

In the context of the present invention, the term “EPIL2 polypeptide” means a polypeptide encoded by the INSL4 gene consisting of: a) from position 115 to position 139 of the amino acid sequence shown in FIG. A chain of the amino acid sequence which is the sequence of the amino acid sequence (including both ends); b) B chain of the amino acid sequence selected from the sequence at positions 18 to 58 (including both ends) of the amino acid sequence shown in FIG. The sequence must be at least from position 24 to 53 (
(Including both ends).

This EPIL2 polypeptide also comprises amino acids 1 to 23 of the amino acid sequence shown in FIG.
(Including both ends). The signal peptide may include a signal peptide having an amino acid sequence selected from the following sequences (including both ends), and the sequence of the signal peptide includes at least the sequence from positions 1 to 17 (including both ends).

In the present invention, the “EPIL3 polypeptide” is coded by the INSL4 gene consisting of the C chain of the amino acid sequence selected from the sequence at positions 54 to 114 (including both ends) of the amino acid sequence shown in FIG. The sequence comprises at least the sequence from position 59 to 114 (inclusive).

In the present invention, the term “bone tissue” preferably refers to a tissue constituting a long, short, or flat cartilage bone. The present invention also relates to a polypeptide according to the present invention, wherein the fetal bone tissue cells are cells of perichondrium.

A subject of the present invention is also an EPIL1, EPIL2 or EPIL3 polypeptide encoded by the INSL4 gene, characterized in that it is expressed in cells of the fetal ligament, in particular the joint ligament.

The present invention also relates to an EP encoded by the INSL4 gene, characterized in that it is involved in the process of differentiation, proliferation and / or regeneration of ligament and / or bone tissue cells.
Also includes IL1, EPIL2 or EPIL3 polypeptides.

An EPIL1, EPIL2 or EPIL3 polypeptide encoded by the INSL4 gene, characterized in that it is involved in the process of bone ossification during cartilage development and / or formation, also forms part of the present invention. I do.

The present invention does not relate to the polypeptide in its native form, ie, the polypeptide was not obtained from its natural environment and was obtained by purification from natural sources,
Or understand that it has been obtained by genetic recombination (possibly glycosylated or non-glycosylated) or alternatively can be obtained by chemical synthesis, in which case it may contain unnatural or modified amino acids Should.

The present invention provides for one or more antibodies against EPIL1, EPIL2 or EPIL3 polypeptides, one of its homologs or variants, or fragments thereof, to EPIL1, EPIL2 in bone tissue and / or ligaments. Alternatively, detection, identification, localization and / or detection of one of the EPIL3 polypeptides or homologs or variants thereof.
Or for use in assays.

The present invention provides for one or more antibodies against EPIL1, EPIL2 or EPIL3 polypeptides, one of its homologs or variants, or fragments thereof, to EPIL1, EPIL2 in bone tissue and / or ligaments. Alternatively, the present invention relates to use of the present invention for diagnosis of a disease associated with abnormal expression of an EPIL3 polypeptide.

[0030] Polypeptides that exhibit certain modifications to the EPIL1, EPIL2 or EPIL3 polypeptides, particularly deletions, truncations, extensions, chimeric fusions and / or mutations, particularly point mutations, are EPIL1 , EPIL2 or EPIL3 polypeptides. Among homologous polypeptides, polypeptides having an amino acid sequence showing a similarity of at least 80%, preferably 90% or more with the amino acid sequence of the polypeptide according to the present invention are preferred.

The term “polypeptide which is a variant of an EPIL1, EPIL2 or EPIL3 polypeptide” refers to truncations, substitutions, deletions of at least one amino acid residue, which may occur naturally in nature, especially in humans. It is intended to mean all mutant polypeptides corresponding to loss and / or addition. These mutant polypeptides are particularly EP
Corresponds to abnormal expression of an IL1, EPIL2 or EPIL3 polypeptide.

The term “a fragment of an EPIL1, EPIL2 or EPIL3 polypeptide, or a homolog or variant thereof” refers to an amino acid comprising at least 5 contiguous amino acids, preferably 10 contiguous amino acids and 15 contiguous amino acids of the polypeptide. Is meant to be a polypeptide of sequence.

[0033] Antibodies that can be used for the detection, identification, localization and / or assay, or diagnosis of the disease include EPIL1, EPIL2 or EPIL3 polypeptides, one of homologs or variants thereof, or fragments thereof. Mono- or polyclonal antibodies or fragments thereof, or chimeric antibodies, characterized in that they can be specifically recognized are preferred.

[0034] Specific polyclonal antibodies can be obtained, for example, from the sera of animals immunized against:-an EPIL1, EPIL2 or EPIL3 polypeptide, one of its homologs or variants, or fragments thereof, Particularly, those produced by genetic recombination or peptide synthesis from a nucleic acid sequence encoding the polypeptide or the amino acid sequence of the polypeptide according to a conventional method.

A specific monoclonal antibody can be obtained according to a conventional hybridoma culture method described in Koeler and Milstein, 1975. The antibody is, for example, a chimeric antibody, a humanized antibody, or a Fab or F (ab ') 2 fragment. According to the invention, the antibody can also be in the form of an immunoconjugate or a labeled antibody in order to obtain a detectable and / or quantifiable signal.

[0036] As used herein, the expression "several antibodies" refers to at least two types of antibodies having different specificities, ie, capable of recognizing and binding to two different epitopes or domains of the polypeptide. It is clearly understood to mean the antibody of Furthermore, according to the present invention, it is preferred to use at least two antibodies with different specificities for the processes, methods and kits of the invention involving said antibodies. In this case, for example, two different antibody labelings or two different revalation systems may be used.

[0037] These antibodies may then be used to express the polypeptide on specific parts of bone and / or ligament tissue, for example by immunofluorescence or gold labeling, or by using enzyme immunoconjugates. Means for analytical or immunohistochemical analysis can be configured.

Since these antibodies allow the detection and quantification of the normal or abnormal specific presence of the polypeptide, especially in bone and / or ligament tissue, the identification and localization of the expression of these polypeptides, Alternatively, it is useful for diagnosing a disease associated with an abnormal presence of the polypeptide.

[0039] More broadly, the antibody provides expression of one of the polypeptides to bone tissue and / or
Alternatively, it can be used to advantage in any situation where qualitative and / or quantitative observations in biological specimens of ligaments are to be made.

[0040] Methods and specific reagents for detecting, identifying, localizing and / or assaying antigen / antibody complexes that can be used in the methods of the invention are well known to those skilled in the art and include, for example, ELISA, RIA. Or immunofluorescence, which may be immunoconjugated, fluorescein-labeled, or capable of specifically recognizing the antibody of the invention, if the antibody is not already immunoconjugated or labeled. Appropriate reagents, such as radiolabeled antibodies, and chromogenic substrates specific for the conjugated enzyme and control reagents for positive, negative, and quantitative controls can be conjugated.

Accordingly, the present invention provides a method for specifically detecting EPIL1, EPIL2 and / or EPIL3 polypeptide or a fragment thereof in a biological sample of bone tissue and / or ligament, comprising the steps of: Methods for identifying, locating and / or assaying, or diagnosing a disease associated with an abnormal presence of the polypeptide in bone tissue and / or in ligaments. Contacting with one or more antibodies against the polypeptide; b) detecting, identifying, localizing and / or assaying the formed antigen / antibody complex.

The present invention also specifically detects and identifies EPIL1, EPIL2 and / or EPIL3 polypeptides or fragments thereof in a biological sample of bone tissue and / or ligament, characterized by comprising the following elements: Kits or packs for locating and / or assaying, or for diagnosing a disease associated with the abnormal presence of the polypeptide in the biological sample: a) one or more of the polypeptides Two or more antibodies; b) if necessary, reagents for constituting a medium suitable for the immunological reaction; c) if necessary, detection of an antigen / antibody complex produced by the immunological reaction; Reagent for identification and / or assay.

The present invention provides a method for detecting, identifying, locating, amplifying and / or assaying a nucleic acid sequence corresponding to an INSL4 gene in bone tissue and / or ligaments, wherein the nucleic acid sequence corresponds to the INSL4 gene. It also relates to the use of oligonucleotide probes or primers capable of hybridizing to

The present invention also provides oligos capable of specifically hybridizing to a nucleic acid sequence corresponding to the INSL4 gene for diagnosis of a disease associated with abnormal expression of the INSL4 gene in bone tissue and / or ligament. It also includes the use of nucleotide probes or primers.

The term “nucleic acid sequence corresponding to the INSL4 gene” refers to the nucleotide sequence of the INSL4 gene shown in FIG. 1 or any nucleotide encoding a polypeptide that is a homolog or variant of an EPIL1, EPIL2 or EPIL3 polypeptide. Sequence, its complementary sequence, the nucleotide sequence of its corresponding RNA, as well as fragments thereof.

The oligonucleotide probe capable of specifically hybridizing with the nucleic acid sequence corresponding to the INSL4 gene may or may not be labeled or immobilized on a solid support by a covalent bond. Methods for labeling or immobilizing such probes are well known to those skilled in the art.

Specific hybridization refers to performing hybridization under stringent conditions, particularly under conditions of temperature and ionic strength, that can maintain hybridization between two DNA fragments that are totally complementary. Means

By way of example, the conditions of high stringency in the hybridization step for defining the nucleotide sequence described above are advantageously as follows. Hybridization was performed with 6 × SSC buffer, 5 × Denhardt's solution, 0.5% SDS and
Performed at a preferred temperature of 65 ° C. in the presence of 100 μg / ml salmon sperm DNA.

1 × SSC corresponds to 0.15 M NaCl and 0.05 M sodium citrate, and 1 × Denhardt's solution corresponds to 0.02% ficoll, 0.02% polyvinylpyrrolidone and 0.02% bovine serum albumin.

The washing step is performed, for example, by using 0.1% SDS in 2 × SSC or 1 × SSC buffer.
It can be performed at 65 ° C. for 5 to 30 minutes. One of skill in the art will adjust the above high stringency hybridization conditions for a given length of polynucleotide to longer or shorter oligonucleotides according to the teachings of Sambrook et al., 1989. .

The above oligonucleotide probes or primers, and generally the nucleic acid sequences according to the invention, will have a length of at least 10 bases and will preferably be fragments of 20 bases or more, preferably 30 bases or more.

Specific detection of a nucleic acid sequence corresponding to the INSL4 gene in a biological sample of bone tissue and / or ligament, which also forms part of the present invention, comprises the following steps: Methods for identifying, and / or assaying, or diagnosing a disease associated with an abnormal presence of the nucleic acid sequence in the biological sample: a) separating DNA from the biological sample to be analyzed Or the RNA of the biological sample
B) specifically amplifying said nucleic acid sequence with at least one primer capable of specifically hybridizing with a nucleic acid sequence corresponding to the INSL4 gene; c) obtaining the amplified product Analyze qualitatively and / or quantitatively.

The present invention also specifically detects, identifies and / or assayes for a nucleic acid sequence corresponding to the INSL4 gene in a biological sample of bone tissue and / or ligament, comprising the following steps: And methods for diagnosing a disease associated with the abnormal presence of the nucleic acid sequence in the biological sample: a) capable of specifically hybridizing to a nucleic acid sequence corresponding to the INSL4 gene. An oligonucleotide probe is included in the biological sample and the biological sample.
Contacting the probe under conditions that allow it to hybridize to DNA, wherein the DNA contained in the biological specimen is, where appropriate, pre-susceptible to hybridization; b). Hybrids formed between the oligonucleotide probe and the DNA of the biological specimen are detected, identified and / or assayed.

The present invention also specifically detects, identifies and / or assayes for a nucleic acid sequence corresponding to the INSL4 gene in a biological sample of bone tissue and / or ligament, which comprises the following steps: Or a method for diagnosing a disease associated with the abnormal presence of the nucleic acid sequence in the biological sample: a) capable of specifically hybridizing to a nucleic acid sequence corresponding to the INSL4 gene Contacting an oligonucleotide probe immobilized on a carrier with the biological sample under conditions that allow hybridization of the probe to DNA of the biological sample, wherein the biological sample is The DNA has been previously amplified, where appropriate, using at least one primer capable of specifically hybridizing to a nucleic acid sequence corresponding to the INSL4 gene, And / or preliminarily susceptible to hybridization; b) combining, if appropriate, the hybrid formed between the oligonucleotide probe immobilized on a carrier and the DNA contained in the biological specimen. Removes the DNA of the biological sample that did not hybridize with the probe, and then contacts a specifically labeled oligonucleotide probe capable of specifically hybridizing with a nucleic acid sequence corresponding to the INSL4 gene. .

What also forms part of the present invention is characterized in that it comprises the following elements:
Abnormal presence of the nucleic acid sequence in a biological sample of bone tissue and / or ligament for specifically detecting, identifying and / or assaying a nucleic acid sequence corresponding to the INSL4 gene, or in the biological sample A kit or pack for diagnosing a disease associated with: a) an oligonucleotide probe capable of specifically hybridizing to a nucleic acid sequence corresponding to the INSL4 gene; b) optionally performing hybridization. Necessary reagents; c) optionally, at least one primer capable of specifically hybridizing with a nucleic acid sequence corresponding to the INSL4 gene, and reagents required for a DNA amplification reaction.

The present invention also specifically detects, identifies and / or tests for a nucleic acid sequence corresponding to the INSL4 gene in a biological sample of bone tissue and / or ligament, characterized in that it comprises: A kit or pack for diagnosing a disease associated with the abnormal presence of the nucleic acid sequence in the biological sample; a) INSL4, which can be supplied in a pre-immobilized state on a carrier. An oligonucleotide probe capable of specifically hybridizing with a nucleic acid sequence corresponding to the gene, a so-called capture probe; b) an oligonucleotide probe which is specifically labeled and capable of specifically hybridizing with a nucleic acid sequence corresponding to the INSL4 gene C) if necessary, specifically hybridized with the nucleic acid sequence corresponding to the INSL4 gene At least one primer that can be formed, and reagents necessary for a DNA amplification reaction.

Also forming part of the present invention is the abnormal development and / or formation of bone in cartilage for diagnosing diseases that result in abnormal proliferation and / or differentiation of ligament and / or bone tissue cells. A method or kit according to the present invention for diagnosing a disease associated with abnormal ossification of and / or diagnosing osteoporosis and / or dysplasia. Dysplasia can in particular include diaphyseal dysplasia or dysplasia or even diaphyseal dysplasia such as diaphyseal hyperplasia or Engelmann disease.

In the present invention, “to diagnose a disease associated with abnormal proliferation and / or differentiation of bone tissue and / or ligament cells, or to abnormally develop and / or abnormal ossification of bone during formation of cartilage. The phrase "for diagnosing an associated disease or for diagnosing osteoporosis and / or for dysplasia" means not only the diagnosis of the disease itself, but also, for example, the disease after treatment. It also implies diagnosis or prognosis of occurrence. Thus, according to the present invention, the method or kit can also be used to evaluate the efficacy of a therapeutic treatment for the disease.

Methods for specific amplification and qualitative and / or quantitative analysis of nucleic acid sequences that can be used in the methods of the invention are well known to those skilled in the art and include, for example, at least one nucleic acid sequence as described above. This is a method including at least one step of so-called PCR (polymerase chain reaction) or PCR-like amplification of a target sequence that may be present using an oligonucleotide primer. The amplified product may be treated with an appropriate restriction enzyme, particularly before detecting or assaying the target product according to the method of the present invention.

The term “PCR-like” is intended to mean any method in which direct or indirect replication of a nucleic acid sequence is performed or the labeling system has already been amplified. These methods are, of course, known. Generally this involves amplification of the DNA by a polymerase;
If the first sample is RNA, reverse transcription should be performed beforehand. Currently,
There are numerous ways to make this amplification possible, for example the so-called NASBA (Nucleic Acid Sequence Based Amplification) (Compton 1991),
TAS (Transcription based Amplification System) (Guatelli
et al., 1990), LCR (Ligase Chain Reaction) (Landegren e
t al., 1988), ERA (Endo Run Amplification), CPR (Cycling Probe Reaction), and SDA (Strand Displacement Amplification)
All are well known to those skilled in the art, for example, by the Displacement Amplification ") method. Also, for example, a nucleic acid to be quantified can be hybridized with the same primer as the target nucleic acid. Nucleic acid quantification methods in the presence that are amplified by PCR-type methods are also known to those skilled in the art.

The amplified fragment can be identified, for example, after agarose or polyacrylamide gel electrophoresis, or after a chromatographic method such as gel filtration or ion exchange chromatography. The specificity of the amplification can be controlled, for example, by molecular hybridization using probes as described above, in particular labeled probes.

The present invention also includes a method of labeling ligament and / or bone tissue cells, especially those derived from the fetus, characterized by comprising the following steps: a) Bone tissue and / or ligament biology Target specimens, EPIL1, EPIL2 and / or
Contacting with one or more antibodies against the EPIL3 polypeptide, wherein the antibodies can be optionally labeled; b) if appropriate, labeled compounds capable of recognizing the antibodies Is used to label the antibody.

Methods for labeling cells with antibodies specific for the compounds of the cells are well known to immunocytochemists or immunohistochemists and will not be described in detail herein. Also encompassed by the present invention is a method of labeling ligament and / or bone tissue cells, especially those of fetal origin, characterized by comprising the following steps: a) Bone tissue and / or ligament ligaments The biological sample is contacted with one or more oligonucleotide probes capable of specifically hybridizing to a nucleic acid sequence corresponding to the INSL4 gene, wherein the probes may be optionally labeled B) if appropriate, labeling the probe with a labeled compound capable of recognizing the probe.

One subject of the present invention is also characterized in that the labeling method according to the present invention is performed.
A method for detecting abnormal ligament and / or bone tissue cells. Another aspect of the present invention relates to an EPIL1, EPIL2 or EPIL3 polypeptide, one of its homologous or mutant polypeptides, or a fragment thereof, a nucleic acid sequence corresponding to the INSL4 gene, or a ligament or bone tissue cell, especially a fetal-derived one. A method of selecting a chemical or biochemical compound capable of altering the differentiation, regeneration and / or proliferation of human and / or animal ligament and / or bone tissue cells, comprising using Also concerns. The cells used may be cells transformed by genetic recombination or selected according to the invention.

Of these methods, the selected compound inhibits or promotes the differentiation, regeneration and / or proliferation of human and / or animal ligament and / or bone tissue cells, especially fetal perichondrocytes. A method characterized in that it can be performed is preferred.

The compound may be capable of binding to, for example, an EPIL1, EPIL2 or EPIL3 polypeptide, one of its homologous or mutant polypeptides, or a fragment thereof, and the compound may, for example, be expressed on cells of the tissue. As an agonist or antagonist ligand for a receptor specific for the polypeptide, in bone tissue and / or
Or its ability to inhibit or enhance the activity of the polypeptide in the ligament.

The compound also has the ability to bind to a nucleic acid sequence corresponding to the INSL4 gene, and to suppress or promote the expression of a gene encoding the polypeptide as an inducer or repressor. Ability.

The compound may also alter the biological activity of the polypeptide expressed by the cell on the cell in vitro or in vivo, particularly the activity of the cell such as differentiation, proliferation or regeneration. You may make choices regarding abilities.

For example, the invention is capable of binding to an EPIL1, EPIL2 or EPIL3 polypeptide, one of its homologous or mutant polypeptides, or a fragment thereof, characterized in that it comprises the following steps: The method includes selecting a compound capable of binding to a nucleic acid sequence corresponding to the INSL4 gene and altering the activity of the polypeptide in bone tissue and / or in ligaments. Contacting a polypeptide or the nucleic acid sequence; b) determining the ability of the compound to bind to the polypeptide or the nucleic acid sequence; c) ligaments and / or potential of the compound selected in step b) Demonstrates altered activity of the polypeptide on bone tissue cells.

For example, in steps b) and / or c) of the method, ligament and / or bone tissue cells transformed by genetic recombination or selected according to the invention could be used.

The present invention also encompasses chemical or biochemical compounds that can be selected by the method according to the invention. The compound that can be selected can be an organic compound, such as a polypeptide or carbohydrate or any other known compound, or has been developed using molecular modeling methods known to those of skill in the art and can be synthesized by chemical or biochemical synthesis. The obtained novel organic compound may be used.

One subject of the invention is also a compound according to the invention as a pharmaceutical product, preferably selected from: a) an EPIL1, EPIL2 or EPIL3 polypeptide, one of its homologous or variant polypeptides, Or fragments thereof, which can be obtained by genetic recombination or chemical synthesis; b) an antibody against the polypeptide described in a) above; c) a ligand of the polypeptide described in a) above. D) a vector containing all or part of the nucleic acid sequence corresponding to the INSL4 gene; e) a marker specific for cells of the target ligament or bone tissue whose alteration is being sought. The vector according to d) above, which has on its surface; f) a sense or antisense capable of specifically hybridizing with a nucleic acid sequence corresponding to the INSL4 gene; Sense nucleic acid sequence.

The EPIL1, EPIL2 or EPIL3 polypeptide described in a) above, or a fragment of a homologous or mutant polypeptide thereof, is preferably characterized in that it is biologically active.

The term “biologically active fragment” refers to EPIL1, EPIL2 or EPIL
3 polypeptide or a fragment of the amino acid sequence of a homologous or variant polypeptide thereof, wherein the fragment exhibits at least one of the activities of said polypeptide, in particular the following activities: Or the ability to alter, particularly suppress or promote the differentiation, proliferation and / or regeneration of bone tissue cells; and / or-Recognized by an antibody specific for an EPIL1, EPIL2 or EPIL3 polypeptide or one of its homologous or mutant polypeptides. Ability to be.

A recombinant EPIL1, EPIL2 or EPIL3 polypeptide, a homologous or mutant polypeptide or a fragment thereof is transformed with a cloning and / or expression vector containing a nucleic acid sequence encoding the polypeptide and the vector. It can be obtained by using a host cell and a method well known to those skilled in the art.

The resulting recombinant polypeptide can be in either glycosylated or non-glycosylated form, and may or may not have a native tertiary structure. These polypeptides can be produced from a nucleic acid sequence encoding the polypeptide according to methods for producing a recombinant polypeptide known to those skilled in the art. In this case, the nucleic acid sequence used is placed under the control of signals that allow its expression in the host cell.

An efficient system for producing a recombinant polypeptide requires a vector, for example, derived from a plasmid or virus, and a compatible host cell. The host cell can be a prokaryotic cell line, such as a bacterium, or a eukaryotic cell line, such as, for example, a yeast, insect, or mammalian cell (such as a CHO cell <Chinese hamster ovary cell> or a mouse cell), or any other cell line. It can be chosen from advantageously available systems. Preferred host cells for expressing the protein of the present invention consist of Chinese hamster ovary cells CHO and 3A-subE cells derived from human placenta.

The vector should include a promoter, translation initiation and termination signals, and appropriate regions for transcriptional regulation. The vector should be able to be stably maintained in the cell and may optionally have specific signals that direct the secretion of the translated protein.

[0079] These various control signals are selected depending on the host cell to be used. For this purpose, the nucleic acid can be inserted into a vector that replicates automatically in the host of choice or into a vector that integrates into the host of choice.

[0080] Such vectors are prepared according to methods currently used by those skilled in the art, and the resulting clones are introduced into appropriate host cells by standard methods, eg, lipofection, electroporation or heat shock. can do.

The nucleotide sequence inserted into the vector as described above is introduced into this cell,
By culturing the cells under conditions that allow replication and / or expression of the transfected nucleotide sequence, a host cell transformed with the above vector can be obtained.

Host cells that can be used for this purpose include, of course, bacterial cells (Olins and Lee,
1993), but also yeast cells (Buckholz, 1993), and animal cells, especially mammalian cell cultures (Edwards and Aruffo, 1993), and especially Chinese hamster ovary cells (CHO), derived from placenta And human cells such as 3A-subE cells, and insect cells (Luckow, 1993), for example, which can utilize the method using baculovirus. Methods used to purify recombinant or synthetic polypeptides are known to those skilled in the art. Recombinant or synthetic polypeptides
Methods such as fractionation, chromatography, or immunoaffinity using specific mono- or polyclonal antibodies can be used alone or in combination to purify.

A preferred variant is a recombinant polypeptide fused to a “carrier” protein
(Complex protein). The advantages of this system include stabilization of the recombinant product and reduced proteolysis, increased solubility during in vitro reconstitution, and / or simplified purification when the fusion partner has affinity for a specific ligand Is possible.

[0084] EPIL1, EPIL2 or EPIL3 polypeptides, homologous or mutant polypeptides thereof, or fragments thereof, can also be obtained by chemical synthesis. Polypeptides obtained by chemical synthesis may contain unnatural amino acids.

The compounds selected as pharmaceutical products include, in particular, antisense oligonucleotides, ie oligonucleotides whose structure ensures inhibition of the expression of the corresponding product by hybridization with the target sequence. it can. INSL
4 Sense oligonucleotides that induce either inhibition or activation of this expression by interaction with proteins involved in the regulation of gene expression may also be mentioned.

The present invention treats diseases that result in abnormal proliferation and / or differentiation of bone tissue and / or ligament cells, particularly diseases that result in abnormal development of cartilage and / or abnormal ossification of bone during formation. And particularly for the treatment of osteoporosis and / or dysplasia.

As the dysplasia, diaphysis dysplasia or dysplasia as well as diaphyseal dysplasia such as diaphyseal hyperplasia or Engelmann disease are particularly preferred. One subject of the present invention is ultimately for the treatment of diseases associated with the regeneration of bone tissue and / or ligament cells, in particular for the treatment of bone tissue and / or ligament damage following traumatic shock. A compound according to the present invention.

The compounds of the present invention as active ingredients in a pharmaceutical product will preferably be in soluble form and mixed with a pharmaceutically acceptable vehicle. Preferably, these compounds will be administered systemically, especially intravenously, intramuscularly, intradermally or orally.

The method of administration, dosage and optimal dosage form generally take into account the establishment of a therapy tailored to the patient, such as, for example, the age or weight of the patient, the severity of the general symptoms, resistance to treatment and side effects. It can be determined according to criteria.

The deficiency or overexpression of a polypeptide according to the invention can generally also be offset by so-called “compensation” therapies which allow the amplification or reduction of the activity of the polypeptide.

[0091] Replacement therapy involves gene therapy, ie, for example, when one of the genes is poorly expressed or is expressed in an abnormal form, and the nucleic acid sequence according to the invention and / or its This can be done by introducing a corresponding gene having elements that allow for in vivo expression.

The principles of gene therapy are known. For example, adenovirus (Perricaudet et
al., 1992), AAV (adenovirus associated virus) (Carter, 1993), retrovirus (Temin, 1986), poxvirus or herpes virus (Epstein et a.
l., 1992). At present, most of these viruses are used in defective form, generally with or without integration into the cell genome. It is also possible to envisage the use of non-viral, ie synthetic, vectors which mimic the viral sequences or in particular consist of naked DNA or RNA according to the method developed by VICAL.

In most cases, it will be necessary to consider targeting elements which ensure specific expression so that the expression region of the polypeptide can be restricted. In some cases, it may be advantageous to have a vector for transient expression, or at least controlled expression that can be blocked when needed.

The compounds according to the invention which can be used as medicinal products are characterized by the uncontrolled (disordered) proliferation or differentiation of cells or bone and / or ligament tissue, or the absence of ligament and / or bone tissue cells, for example following trauma. Or a new therapeutic method for a disease associated with deficiency.

[0095] Other features and advantages of the present invention will become apparent in the remainder of the specification, with the examples and figures described below.

[0096]

【Example】

Materials and Methods A) Experimental Subjects Morphologically normal embryos at the age of 3 to 8 weeks (post-ovulation) were obtained at Brussels Hospital in Paris after legal abortion with mifepristone (RU 486). Was. Complete independence was observed between the hospital's medical management and the study groups. He never sought the consent of his mother before abortion was decided and implemented. Consent was obtained only after completing medical abortion and after explaining the purpose of the study. This procedure has been approved by the Ethics Committee of the Neckar Hospital for Sick Children (Paris). Embryos were separated from trophoblasts by dissection under a microscope. The stage of development of individual embryos was estimated using the Carnegie classification established by Olahillie (R. O'Rahilly et al., 1987 and 1994). Microdissected embryos were frozen and stored at -80 ° C.
Frozen sections (12 μm) pretreated slides (Probon Plus slides, Microm
France). Sections (dissected specimens) were placed in 0.1 M phosphate buffer (pH 7.4)
Fix in a solution of medium paraformaldehyde for 30 minutes, rinse once in PBS (phosphate-buffered saline) and briefly in water, then use a series of ethanol solutions of different concentrations
, 75%, 95%). Sections were then air dried and stored at -80 ° C to preserve mRNA.

B) Hybridization Probe A 60-mer oligonucleotide probe was synthesized and purified (DNAgency, Malvern, PA). Using a deoxyribonucleotide terminal transferase (Gibco BRL, Grand Island, NY), the probe was probed with [α- ³⁵ S] dATP (NEN, Boston, MA) for about 7 minutes.
3′-labeled at a specific activity of × 10 ⁸ cpm / mg. Prior to using this oligonucleotide probe on a Biospin column (Bio-Rad Laboratories, Hercules, CA)
And purified. The sequence of the antisense INSL4 probe is as follows (SEQ ID NO: 2): 5'-CTGGGGTGGTGGTGAATGTCTTCTCAGGCATGGGGCAATATGACAGCAAGTGTTTTCCAA-3 ', located in exon 1. The sequence of the antisense IGF-II probe is as follows (SEQ ID NO: 3): 5′-AGCCAATCGATTTTGTACATGTTTGAAGATGCTGCTGTGCTTCCTCAGCCCGATGGAGGG-3 ′, IGF-
Located at exon 7 in the consensus coding region of the II mRNA. A sense probe was used as a control.

C) In situ hybridization Experimental protocol (M. Abitbol et al., 1993 and M. Gerard et al, m 1995)
Are formamide 50%, 4 × SSC (standard salt-citrate buffer), 1 × Denhardt's solution, 0.25 mg / ml yeast tRNA, 0.25 mg / ml herring sperm [lucuna], 0.25 mg / ml polyA
, 10% dextran sulfate, 100 mM dithiothreitol (DTT) and a specific activity of 6 ×
Of ^{10 6 cpm / ml [- 35} S] containing the hybridization process using a solution containing dATP labeled probe. 100 μl of this hybridization solution was placed on each section. The sections were then covered with parafilm and incubated in a humidity chamber at 43 ° C. for 20 hours. After hybridization, slides were washed with 1 × SS containing 10 mM DTT.
Twenty-five minutes at 55 ° C. in C and twice for 15 minutes at room temperature in 0.5 × SSC buffer containing 10 mM DTT. Sections were then immersed in water and dehydrated using a series of ethanol solutions of different concentrations, and then on Amersham Hyperfilm Betamax X-ray film for 1 week, Kodak NTB2 (Eastman Kodak, Rochester, NY) 1-2 months in photographic emulsion Exposure was at 4 ° C. Thereafter, the sections were developed and stained with toluidine blue. The results are analyzed by phase contrast microscopy.

Example 1 In Situ Hybridization of Placenta and Embryonic Membrane To demonstrate the specificity of the oligonucleotide, total RNA from trophoblast tissue was
Northern blot analysis was performed using μg. The INSL4 transcript was detected to be 0.7 kilobases (kb) long using both oligonucleotide and cDNA probes. This length is based on INSL4 published by Chassin et al. (1995).
Matches the length of the transcript. For the IGF-II gene, a major transcript of 6.0 kilobases and another transcript of 2-5 kilobases were detected. These are 1.8, 2.0, 2.8,
It confirms the results of Han et al. (1988) who identified multiple IGF-II transcripts with estimated sublengths of 3.0, 4.0, 4.8 and 6.2 kb, with the highest abundance of the last. These results indicate the specificity of the INSL4 and IGF-II oligomers selected for human INSL4 and IGF-II mRNA, respectively. Sense INSL4 and IGF-
In situ hybridization performed with the II oligonucleotide resulted in only a broad array of lines corresponding to background noise, thus demonstrating the specificity of the positive hybridization obtained with the antisense oligonucleotide.

Low levels of INSL4 gene expression were detected in the early placenta at 25 days of age, and abundant levels of INSL4 mRNA were identified in the 14-week-old placenta. The INSL4 gene is expressed in the syncytiotrophoblast layer and, to a lesser extent, in the cytotrophoblast. Central cells of chorionic mesoderm and umbilical cord cells express the INSL4 gene even weaker (FIGS. 2A, 2B and 2C). No signal was detected in the amniotic membrane or yolk sac (results not shown).

IGF-II mRNA was expressed on intermediate trophoblast columns at 20 days and 14 weeks of development.
It was abundantly expressed in the hoblast column) and in the villous stroma, but could not be detected in sections of the syncytiotrophoblast layer (FIGS. 2D, 2E and 2F). Embryo membrane
At (day 20), abundant amounts of IGF-II mRNA were identified in amniotic cells and outer cells of the yolk sac (results not shown). These results are summarized in Table 1 below.

[0102]

[Table 1]

Symbols in Table 1: Nd: not measured; Neg .: negative hybridization signal; +: weak hybridization signal; ++: moderate hybridization signal; +++: strong hybridization signal; +++: very strong Hybridization signal; signal observed after 6 weeks of exposure.

The INSL4 gene is more highly expressed in syncytiotrophoblasts than in cytotrophoblasts. These results were based on RT-PCR (polymerase chain reaction amplification using reverse transcriptase) performed on total RNA from syncytiotrophoblast cells and cytotrophoblast cells (D. Bellet et
al., 1997). Notably, IGF-II mRNA is more abundant than INSL4 mRNA in adjacent sections tested using INSL4 oligonucleotide probes. Expression of the IGF-II gene in human embryonic placentas at 25 days, 40 days and 14 weeks of age is detected in cells at the center of chorionic mesoderm as well as in cells of the intermediate cytotrophoblast or the villus trophoblast. Located in the cell. IGF-II transcripts are not detected in the syncytiotrophoblast layer for the developmental stages examined. Similar findings have been reported, for example, by VKM Han (VKM Han et al., 1996). Therefore, it is highly probable that these two genes play different roles in the development of trophoblasts.

Example 2 In Situ Hybridization of Embryonic Tissue Unlabeled INSL4 oligonucleotide probes were detected in 20 or 30 day old embryonic tissues. This suggests that the INSL4 gene is not expressed at this early stage or that the level of transcription is too low to be detected by in situ hybridization. Conversely, the IGF-II gene was strongly expressed at this early embryonic stage. Expression of the IGF-II gene has been demonstrated in several tissues from mesoderm and endoderm (FIGS. 3A-3H). These results are described in the previous report (VKM Han et
al., 1987a and 1987b; JE Lee et al., 1990). The major site of IGF-II expression is located in the liver for all stages examined.

[0106] Sections of 8 week old fetal tissue were also hybridized using an INSL4 oligonucleotide probe. Surprisingly, INSL4 mRNA showed a specific spatial distribution. Specifically, the majority of INSL4 transcripts are located in the perichondrium of the limbs, ribs and vertebrae. INSL4 mRNA is also very abundant in joint ligaments (FIGS. 4A-4F). Conversely, at this developmental stage, no label was detected in the liver, lung, heart, thymus, stomach, muscle or small intestine. Table 2 below summarizes the results according to the cell types identified in the sections of fetal tissue tested.

[0107]

[Table 2]

Symbols in Table 2: Nd: not measured; Neg .: negative hybridization signal; +: weak hybridization signal; ++: moderate hybridization signal; +++: strong hybridization signal; +++: very strong Hybridization signal; signal observed after 6 weeks of exposure.

[0110] Sections of embryonic tissue hybridized with the same INSL4 and IGF-II oligonucleotide probes were obtained from IGF-II transcripts (VRKs) located in most embryonic tissues.
Unlike Han et al., 1987a and 1987b; JE Lee et al., 1990), INSL4
The gene shows that it is only slightly expressed in these tissues. Specifically, in sections of 8-week-old human embryo tissue, INSL4 transcripts are located only in perichondrium and ligament cells. At this stage of development, cartilage has begun in all bones of the lower limbs.

It should be noted that the INSL4 gene is a member of the superfamily of insulin-like genes, and that other members of this family encode polypeptides that regulate both growth and development in many tissues. . In particular, insulin-like growth factor (IG
F) plays an important role in regulating bone formation (B. Bhaumic et al., 1993; R. Malpe
et al., 1997, and S. Mohan et al., 1991). In this regard, the specific expression of the INSL4 gene during human embryonic development is related to the development of the bone and ligaments during which it is forming, both in terms of timing and its highly restricted tissue distribution within the embryo. Strongly suggest that they are almost certainly involved in the various stages of

Literature Abitbol M., et al., 1993, Basal nuclear magnocelluraris and hippocampus are major sites of FMR-1 expression during human embryonic development, preliminary report, CR Acad.
i. Paris 318: 57-66; Adam, IM et al., 1993, J. Biol. Chem. 268: 26668-26672; Baker J., et al. 1993, Insulin-like proliferation in embryo and postnatal growth. Role of factors, Cell. 75: 73-82; Bellet D., et al., 1997, Identification of pro-EPIL and EPIL peptides translated from insulin-like 4 (INSL-4) mRNA in human placenta, J Clin. Endocrinol Metab. 82:
3169-3172 ・ Bhaumic B., 1993, Insulin-like growth factor (IGF) binding protein and insulin-like growth factor secretion by cultured chondrocytes: identification, characterization and ontogeny during cell differentiation, Regulatory Peptides 48: 113-122・ Bogic, LV et al., 1995, J. Clin. Endocrinol.Metab. 80: 130-137; ・ Buckholz, RG, 1993, Curr. Op.Biotechnology 4: 538-542; ・ Burn, J., et. al., 1995, Using Human Embryos in Developmental Studies, Nature Genet. 11
: Carter, BJ, 1993, Curr. Op. Biotechnology 3: 533-539; Chassin D., et al., 1995, a new member of the insulin gene superfamily (INSL4) expressed in human placenta. Cloning, Genomics 29: 465-470; Compton, J., et al., 1991, Nature, 350: 91-92; DeChiara TM., Et al., 1990, Insulin-like cleavage by targeting Growth factor
Growth deletion phenotype in heterozygous mice with II, Nature 345: 78-80; Edwards, CP, et al., 1993, Curr.Op.Biotechnology 4: 558-563; Epstein, A., 1992, Medicine / Sciences 8: 902-911; Gerard M., et al., 1995, Pax gene expression during human embryonic development, preliminary report, Compte
Rendu Acad. Sci. Paris 318: 57-66; Guatelli JC, et al., 1990, Proc. Natl. Acad. Sci. USA, 87: 1874-1878
Han VKM, et al., 1987a, Identification of human fetal somatomedin / insulin-like growth factor immunoreactive cells, Pediatrics Research 22: 245-249; Han VKM, et al., 1987b, Human fetal somatomedin ( Insulin-like growth factor)
Cell localization of messenger RNA, Science 236: 193-197; Han VKM et al., 1988, Expression of human fetal somatomedin (insulin-like growth factor) messenger ribonucleic acid: identification, characterization and tissue distribution, Clin. Endoc
rinol. Metab. 66: 422-429; Han VKM et al, 1996, insulin-like growth factor of human placenta at the fetal-maternal interface (IGF
) And expression of IGF binding protein (IGFBP), J. Clin. Endocrinol. Metab.
81: 2680-2693; Hansell DJ, et al., 1991, Human relaxin in decidua, trophoblasts and prostate
H1 gene expression, J. Clin. Endocrinol.Metab. 72: 899-904; Koeler and Milstein, 1975, Nature 256: 495-497; Landegren, U. et al., 1988, Science 241: 1077-1080. Lee JE, et al., 1990, Patterns of insulin-like growth factor II gene expression during early mouse embryogenesis, Development 110: 151-159; Le Roith D., 1997, Insulin-like growth factor, N. Engl . J. Med. 336: 633-64
Liu JP, et al., 1993, Insulin-like growth factor I (Igf-1) and type 1 IGF
Mouse with a null mutation in the gene encoding the receptor (Igf1r), Cell. 75:59.
-72 ・ Malpe R., 1997, Insulin-like growth factor (IGF) in conditioned medium of normal human bone cells
I, -II, IGF binding protein (IGFBP) -3, -4 and -5 concentrations are skeletal site dependent,
J. Bone Mineral Res. 12: 423-430; Mohan S., et al., 1991, bone growth factor, Clin. Orthop. Rel. Res. 263: 30-48
Olins, PO, and Lee, 1993, Curr. Op. Biotechnology 4: 520-525; O'Rahilly R., et al., 1987, Developmental stages of human embryos and a review of Streeter's 'Horizon' and Carnegie Survey of the Collection, Carnegie Institute, Washington, Publication No. 637; O'Rahilly R., et al., 1994, Embryonic human brain, developmental stage illustration, Wiley-Liss, In
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Specific and comprehensive relationships, TIG in press; Perricaudet, M. et al., 1992, La Recherche 23: 471-473; Sambrook, J., et al., 1989, Molecular cloning: Manual in laboratory , New York, Cold Spring Harbor, Cold Spring Harbor Laboratory Pr
ess; • Strachan T., et al., 1997, New dimension of the human gene project: Towards a comprehensive expression map, Nature Genet. 16: 126-132; • Tashims LS et al., 1995, Human Leydig The insulin-like (hLEY IL) gene is expressed in the corpus luteum and trophoblast, J. Clin. Endocrinol Metab. 80: 707-710; Walker, GT et al., (1992), Nucleic Acids Res. 20: 1691- 1696

[Sequence list]

[Brief description of the drawings]

FIG. 1 shows the nucleic acid sequence of the cDNA of the INSL4 gene and the amino acid sequence of the EPIL polypeptide encoded by this sequence.

FIG. 2A, 2B, 2C, 2D, 2E and 2F: INSL4 and IGF-II m in 14-week-old human placenta
Comparison of RNA distribution;-Brightfield (Fig. 2A) and darkfield (Fig. 2B) observations of the same incision of tissue hybridized with the antisense INSL4 probe;-Magnification of the incision observed in brightfield. Figure (FIG. 2C);-Observation of the same incision of the tissue hybridized with the antisense IGF-II probe in the bright field (FIG. 2D) and dark field (FIG. 2E);-The incision specimen observed in the bright field S: syncytiotrophoblast cell layer; c: trophoblast cell layer; m: mesenchyme; scale lines represent 295 μm for FIGS. 2A, 2B, 2D and 2E; FIG. 2F represents 15 μm.

FIG. 3A, 3B, 3C, 3D, 3E, 3F, 3G and 3H: 25-day-old (FIGS. 3A and 3B) obtained by in situ hybridization using a ³⁵ S-labeled oligonucleotide probe.
In human embryos at 30 days of age (Figures 3C, 3D, 3E and 3F), or 45 days of age (Figures 3G and 3H).
Cell distribution of IGF-II mRNA. Micrographs of brightfield (left column) and darkfield (right column) of the same tissue incision specimen;-axial incision specimen at the level of the "brain brain" (Figures 3A and 3B); nt: neural tube; ov : Longitudinal abdominal incision (FIGS. 3C and 3D); nt: neural tube; m: mesonephros;-saggital incision at gill arch (ba) level (FIGS. 3E and 3F) A longitudinal incision of the vertebral body (vb) (FIGS. 3G and 3H); the scale lines represent 295 μm for FIGS. 3A, 3B, 3C and 3D and 150 μm for all remaining pictures.

FIGS. 4A, 4B, 4C, 4D, 4E and 4F: In situ hybridization performed on an 8-week-old human fetus using an INSL4 oligonucleotide probe. Brightfield (left column) and darkfield (right column) micrographs of the same tissue dissection specimen:-Dissection specimen of the distal part of the femur in the hip joint that occurred at the level of the "rhombic brain" (Figure 4A)
And 4B); f: femur; l: ligament;-longitudinal incision of the spine (FIGS. 4C and 4D); INSL4 mRNA is located in the intervertebral disc and in the intervertebral ligament; vb: vertebral body; -Ligament;-Transverse incision of ribs (Figs. 4E and 4F); pc: Perichondrium; r: Ribs; scale lines represent 295 [mu] m for Figs. 4A, 4B, 4C and 4D, and for Figs. 4E and 4F. Represents 150 μm.

[Procedural Amendment] Submission of translation of Article 34 Amendment of the Patent Cooperation Treaty

[Submission date] March 16, 2000 (2000.3.16)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Claims

[Correction method] Change

[Correction contents]

[Claims]

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61K 48/00 C07H 21/00 A61P 19/00 C07K 14/64 C07H 21/00 16/26 C07K 14/64 C12P 21/02 16/26 C12Q 1/68 A C12N 5/10 G01N 33/53 D C12P 21/02 33/569 C12Q 1/68 33/68 G01N 33/53 C12N 15/00 ZNAA 33/569 A61K 37 / 02 33/68 C12N 5/00 B

Claims (40)

[Claims] 1. The INSL4 gene, which is expressed in fetal bone tissue and / or ligament cells. 2. The EPIL1, EPIL2 or EPIL3 polypeptide encoded by the INSL4 gene according to claim 1, which is expressed in fetal bone tissue cells. 3. The method according to claim 1, wherein the fetal bone tissue cells are perichondrium cells.
The polypeptide according to claim 2. 4. The method according to claim 1, which is expressed in fetal ligament cells.
An EPIL1, EPIL2 or EPIL3 polypeptide encoded by the described INSL4 gene. 5. The polypeptide according to claim 2, which is involved in a process of differentiation of a ligament and / or a bone tissue cell. 6. The polypeptide according to claim 2, which is involved in a process of ligament and / or bone tissue cell proliferation. 7. The polypeptide according to claim 2, which is involved in a process of regenerating ligament and / or bone tissue cells. 8. It is involved in the process of cartilage development of the forming bone.
A polypeptide according to any one of claims 2 to 7. 9. The polypeptide according to claim 2, which is involved in the process of ossification of the bone during formation. 10. EPIL1, EPIL2 or EP in bone tissue and / or ligament
EPIL1 according to any of claims 2 to 9 for detecting, identifying, locating and / or assaying an IL3 polypeptide or one of its homologues or variants.
Use of one or more antibodies against one of the EPIL2 or EPIL3 polypeptides, homologs or variants thereof, or fragments thereof. 11. EPIL1, EPIL2 or EPIL1 in bone tissue and / or ligaments
10. An EPIL1, EPIL2 or EPIL3 polypeptide according to claim 2, for diagnosing a disease associated with abnormal expression of the EPIL3 polypeptide, one of homologs or variants thereof, or a fragment thereof. Use of one or more antibodies. 12. Use of an oligonucleotide probe or primer for detecting, identifying, locating, amplifying and / or assaying a nucleic acid sequence corresponding to the INSL4 gene according to claim 1 in bone tissue and / or ligament. 13. A method for diagnosing a disease associated with abnormal expression of the INSL4 gene in bone tissue and / or ligament, specifically hybridizing with the nucleic acid sequence corresponding to the INSL4 gene according to claim 1. Use of oligonucleotide probes or primers that can. 14. The EPIL1, EPIL2 or EPIL3 polypeptide according to claim 2 or a homologue thereof in a biological sample of bone tissue and / or ligament, characterized by comprising the following steps: For specifically detecting, identifying, locating and / or assaying one of the substances or variants, or in bone tissue and / or
Or a method for diagnosing a disease associated with an abnormal presence of said polypeptide in a ligament: a) contacting said biological specimen with one or more antibodies against said polypeptide or a fragment thereof; b) The formed antigen / antibody complex is detected, identified, localized and / or assayed. 15. The EPIL1, EPIL2 or EPIL3 polypeptide according to any one of claims 2 to 9 in a biological sample of bone tissue and / or ligament, or a homolog thereof, comprising the following elements: Kit or pack for specifically detecting, identifying, locating and / or assaying for a product or variant, or for diagnosing a disease associated with the abnormal presence of the polypeptide in the biological sample A) one or more antibodies against the polypeptide or a fragment thereof; b) optionally, reagents for constituting a medium suitable for an immunological reaction; c) optionally, immunology. Reagent for detecting, identifying and / or assaying the antigen / antibody complex generated by the reactive reaction. 16. A method for specifically detecting, identifying and / or detecting a nucleic acid sequence corresponding to the INSL4 gene according to claim 1 in a biological sample of bone tissue and / or ligament, comprising the steps of: Or a method for assaying or diagnosing a disease associated with an abnormal presence of the nucleic acid sequence in the biological sample: a) isolating DNA from the biological sample to be analyzed or RNA of biological specimen
B) specifically amplifying said nucleic acid sequence with at least one primer capable of specifically hybridizing with a nucleic acid sequence corresponding to the INSL4 gene; c) obtaining the amplified product Analyze qualitatively and / or quantitatively. 17. A method for specifically detecting, identifying and / or detecting a nucleic acid sequence corresponding to the INSL4 gene according to claim 1 in a biological sample of bone tissue and / or ligament, comprising the steps of: Or a method for assaying or diagnosing a disease associated with the abnormal presence of the nucleic acid sequence in the biological sample: a) capable of specifically hybridizing to a nucleic acid sequence corresponding to the INSL4 gene An oligonucleotide probe is included in the biological sample and the biological sample.
Contacting the probe under conditions that allow it to hybridize to DNA, wherein the DNA contained in the biological specimen is, where appropriate, pre-susceptible to hybridization; b). Hybrids formed between the oligonucleotide probe and the DNA of the biological specimen are detected, identified and / or assayed. 18. A nucleic acid sequence corresponding to the INSL4 gene according to claim 1 in a biological sample of bone tissue and / or ligament, which comprises the following steps: Or a method for assaying or diagnosing a disease associated with the abnormal presence of the nucleic acid sequence in the biological sample: a) capable of specifically hybridizing to a nucleic acid sequence corresponding to the INSL4 gene Contacting an oligonucleotide probe immobilized on a carrier with the biological sample under conditions that allow hybridization of the probe to DNA of the biological sample, wherein the biological sample is The DNA has been previously amplified, where appropriate, using at least one primer capable of specifically hybridizing to a nucleic acid sequence corresponding to the INSL4 gene; and B) hybridizing between the oligonucleotide probe immobilized on a carrier and the DNA contained in the biological sample, if appropriate, After removing the DNA of the biological sample that has not hybridized to the probe, it is contacted with a specifically labeled oligonucleotide probe capable of specifically hybridizing to the nucleic acid sequence corresponding to the INSL4 gene. 19. A nucleic acid sequence corresponding to the INSL4 gene according to claim 1 in a biological sample of bone tissue and / or ligament, which comprises the following elements: Or a kit or pack for assaying or for diagnosing a disease associated with the abnormal presence of the nucleic acid sequence in the biological specimen: a) specifically hybridizing with a nucleic acid sequence corresponding to the INSL4 gene. An oligonucleotide probe capable of performing the hybridization; b) optionally, a reagent necessary for performing hybridization; c) optionally, at least one kind capable of specifically hybridizing with a nucleic acid sequence corresponding to the INSL4 gene. Primers and reagents required for the DNA amplification reaction. 20. The nucleic acid sequence corresponding to the INSL4 gene according to claim 1 in a biological specimen of bone tissue and / or ligament, which comprises the following elements: Or a kit or pack for assaying or diagnosing a disease associated with the abnormal presence of the nucleic acid sequence in the biological specimen; a) the INSL4 gene, which can be supplied pre-fixed to a carrier An oligonucleotide probe capable of specifically hybridizing with a nucleic acid sequence corresponding to the so-called capture probe; b) an oligonucleotide which is capable of specifically hybridizing with a nucleic acid sequence corresponding to the INSL4 gene, particularly a labeled oligonucleotide. Nucleotide probes, so-called revelation probes; c) if necessary, hybridize specifically with the nucleic acid sequence corresponding to the INSL4 gene. At least one primer that can be used as well as reagents necessary for a DNA amplification reaction. 21. The method or kit according to any one of claims 14 to 20, for diagnosing a disease associated with abnormal proliferation and / or differentiation of ligament and / or bone tissue cells. 22. The method or kit according to claim 21, for diagnosing a disease associated with abnormal cartilage development and / or abnormal ossification of the forming bone. 23. A method for diagnosing osteoporosis and / or dysplasia.
21. The method or kit according to any one of 14 to 20. 24. A method for labeling ligament and / or bone tissue cells, comprising the steps of: a) subjecting a biological sample of bone tissue and / or ligament to a biological sample of any of claims 2 to 9; One or two of the EPIL1, EPIL2 and / or EPIL3 polypeptides described in
Contacting with one or more antibodies, wherein the antibodies may be optionally labeled; b) labeling the antibodies with labeled compounds capable of recognizing the antibodies, where appropriate . 25. A method for labeling ligament and / or bone tissue cells, comprising the steps of: a) applying a biological specimen of bone tissue and / or ligament to the INSL4 gene according to claim 1; Contacting with one or more oligonucleotide probes capable of specifically hybridizing to the corresponding nucleic acid sequence, wherein said probes may be optionally labeled; b) where appropriate, The probe is labeled with a labeled compound that can recognize the probe. 26. The ligament and / or bone tissue cell is a fetal cell.
26. The cell labeling method according to any one of 24 or 25. 27. A method for detecting abnormal ligament and / or bone tissue cells, comprising performing the method according to claim 24. 28. An EPIL1, EPIL2 or EPIL3 polypeptide according to any of claims 2 to 9, one of its homologous or mutant polypeptides or fragments thereof, a nucleic acid sequence corresponding to the INSL4 gene, or a ligament or A chemical or biological agent capable of altering the differentiation, regeneration and / or proliferation of human and / or animal ligaments and / or bone tissue cells, characterized in that it uses bone tissue cells, especially those of fetal origin. How to select a chemical compound. 29. The selection according to claim 28, wherein the selected compound is capable of inhibiting the differentiation, regeneration and / or proliferation of human and / or animal ligament and / or bone tissue cells. Method. 30. The selection according to claim 28, characterized in that the selected compound is capable of promoting the differentiation, regeneration and / or proliferation of human and / or animal ligament and / or bone tissue cells. Method. 31. The selection method according to claim 28, wherein said cells are fetal cells. 32. The fetal cell is a perichondrial cell.
31. The selection method described in 31. 33. A chemical or biochemical compound which can be selected by the method according to claim 28. 34. The compound according to claim 33 as a pharmaceutical product. 35. The method as claimed in claim 33, wherein the material is selected from the following.
A) an EPIL1, EPIL2 or EPIL3 polypeptide, one of its homologous or mutant polypeptides, or a fragment thereof, which can be obtained by genetic recombination or chemical synthesis; b) an antibody against the polypeptide according to a); c) a chemical or biochemical compound characterized by being a ligand for the polypeptide according to a) above; d) all or one of the nucleic acid sequences corresponding to the INSL4 gene A) a vector according to d) above, which has on its surface a marker specific to the cells of the target ligament or bone tissue whose activity is to be altered; f) a nucleic acid sequence corresponding to the INSL4 gene A sense or antisense nucleic acid sequence capable of specifically hybridizing to 36. The compound according to claim 34 or 35 for treating a disease associated with abnormal proliferation and / or differentiation of bone tissue and / or ligament cells. 37. A compound according to claim 36 for treating a disease which results in abnormal cartilage development and / or abnormal ossification of the forming bone. 38. A method for treating osteoporosis and / or dysplasia.
36. The compound according to any of 34 or 35. 39. A compound according to any one of claims 34 or 35 for use in the treatment of a disease involving bone tissue and / or ligament regeneration. 40. The compound of claim 39, wherein said treatment is directed at repairing bone tissue and / or ligament damage following traumatic shock.