JP2016026486A - Ossification of spine ligament model non-human animal, diabetes model non-human animal, ossification of spine ligament model cell, diabetes model cell, screening method, detection method of ossification of spine ligament lesion cell, ossification of spine ligament diagnostic kit and ossification of spine ligament therapeutic agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an ossification of spine ligament model non-human animal that becomes a model of ossification of spine ligament.SOLUTION: (1) An ossification of spine ligament model non-human animal is characterized in that a function of CXCL7 genes is lost or suppressed, or an expression of miRNA-340 is enhanced. (2) The ossification of spine ligament model non-human animal is characterized in that the loss or suppression of the function of the CXCL7 genes is caused by the deficiency, insertion, addition or replacement of a part or the whole of the CXCL7 genes or the expression control region. (3) The ossification of spine ligament model non-human animal is characterized in that the enhancement of the expression of the miRNA-340 is caused by the introduction of the miRNA-340.SELECTED DRAWING: None

Description

  The present invention relates to a spinal ligament ossification model non-human animal, a diabetes model non-human animal, a spinal ligament ossification model cell, a diabetes model cell, a screening method, a detection method of a spinal ligament ossification lesion cell, a spinal ligament ossification The present invention relates to a symptom diagnosis kit and a spinal ligament ossification treatment agent.

Spinal ligament ossification (OSL) is classified into posterior longitudinal ligament ossification (OPLL), yellow ligament ossification, and anterior longitudinal ligament ossification depending on the ossified ligament.
Posterior longitudinal ligament ossification (OPLL) is a spinal canal stenosis caused by ossification of the posterior longitudinal ligament that connects the posterior margin of the vertebral body and runs almost the entire length of the vertebral column. It is a disease that causes The posterior longitudinal ligament ossification (OPLL) is a serious disease accompanied by limb paralysis because the onset is the cervical spine. It is most common in the cervical spine but also occurs in the thoracic and lumbar vertebrae.
Yellow ligament ossification is a disease in which the yellow ligament that exists behind the spinal cord and connects between the vertebral vertebrae becomes ossified, resulting in spinal cord paralysis, and is common in the thoracic vertebra. In patients with posterior longitudinal ligament ossification, ankylosing vertebral hyperplasia that causes ossification of the spinal ligament extensively, mainly in ossification of the anterior longitudinal ligament, is associated with approximately 40%, and ossification of the ligamentum flavum or supraspinal ligament There are also many cases, and there is also an idea that this is regarded as a partial disease of ossification of the spinal ligament.

Conventionally, as a treatment for posterior longitudinal ligament ossification, anterior gradual fixation from the front, laminectomy from the rear, and spinal canal expansion are performed. In addition, laminectomy is performed as a treatment for ossification of the ligamentum flavum. As a prognosis of these diseases, most spinal paralysis is progressive, and minor trauma such as falls may suddenly cause paralysis or hate. If paralysis is left untreated for a long period of time, it will become all-round spinal cord paralysis, and it is difficult to recover even after surgery.
Thus, the present condition is that the effective treatment method is not established for spinal ligament ossification.

The inventors have so far examined the expression state of proteins and the like in patients with spinal ligament ossification, especially posterior longitudinal ligament ossification. As a result, peptide fragments derived from pro-platelet basic protein (chemokine (CXC motif) ligand 7) CXCL7, one of the chemokines present in the plasma of healthy individuals, in the plasma of patients with vertebral ligament ossification Was found to be deficient (Patent Document 1).
However, it has not been demonstrated so far whether the relationship between CXCL7 deficiency and the development of posterior longitudinal ligament ossification, that is, whether CXCL7 deficiency causes ossification of the posterior longitudinal ligament. In addition, there have been no reports on non-human animals that are models for the development of posterior longitudinal ligament ossification.

JP 2011-97867 A

  The present invention has been made in view of the above circumstances, and spinal ligament ossification model non-human animal, diabetes model non-human animal, spinal ligament ossification model cell, diabetes model cell, the non-human animal, or spinal ligament bone It is an object of the present invention to provide a screening method for a prophylactic or therapeutic agent for spinal ligament ossification using keratosis model cells. Another object of the present invention is to provide a method for detecting vertebral ligament ossification lesion cells using a novel marker, a kit for diagnosing spinal ligament ossification, and a therapeutic agent for spinal ligament ossification.

  As a result of earnest research to solve the above problems, the present inventor has found that a mouse deficient in a part of the CXCL7 gene sequence on the chromosome has a phenotype characteristic of vertebral ligament ossification. I found it. In addition, the mice were found to have a phenotype characteristic of diabetes. Moreover, it discovered that the cell by which the expression of CXCL7 was suppressed has a phenotype characteristic of spinal ligament ossification. Furthermore, the present inventor has found that the expression level of miRNA-340 in blood is significantly increased in patients with posterior longitudinal ligament ossification, and by targeting this molecule, vertebral ligament ossification lesion cells It has been found that it is possible to detect.

  That is, the present invention provides a spinal ligament ossification model non-human animal, a diabetes model non-human animal, a spinal ligament ossification model cell, a diabetes model cell, a screening method, a spinal ligament ossification lesion cell having the following characteristics: The present invention provides a detection method, a spinal ligament ossification diagnosis kit, and a spinal ligament ossification treatment agent.

(1) A spinal ligament ossification model non-human animal, wherein the function of the CXCL7 gene is deficient or suppressed, or the expression of miRNA-340 is enhanced.
(2) The spinal ligament bone according to (1), wherein the functional deficiency or suppression of the CXCL7 gene is due to deletion, insertion, addition, or substitution of a part or all of the CXCL7 gene or an expression control region thereof. A non-human animal model.
(3) The spinal ligament ossification model non-human animal according to (1) or (2), wherein the CXCL7 gene is a gene encoding the amino acid sequence represented by SEQ ID NO: 3.
(4) The spinal ligament ossification model non-human animal according to (1), wherein the enhanced expression of miRNA-340 is due to the introduction of miRNA-340.
(5) The spinal ligament ossification model non-human animal according to (1) or (2) above, wherein the miRNA-340 is a polynucleotide comprising the RNA sequence represented by SEQ ID NO: 6.
(6) A diabetic model non-human animal, wherein the function of the CXCL7 gene is deficient or suppressed, or the expression of miRNA340 is enhanced.
(7) A spinal ligament ossification model cell, wherein the function of the CXCL7 gene is deficient or suppressed, or the expression of miRNA-340 is enhanced.
(8) The spinal ligament ossification model cell according to (7), which is a cell of parallel fibrous connective tissue.
(9) A diabetes model cell, wherein the function of the CXCL7 gene is deficient or suppressed, or the expression of miRNA-340 is enhanced.
(10) The spinal ligament ossification model non-human animal according to any one of (1) to (5), a cell obtained from the spinal ligament ossification model non-human animal, or the (7 ) Or (8), a test substance is administered to the spinal ligament ossification model cell according to (8).
(11) The prophylactic agent for spinal ligament ossification according to (10), wherein after administering the test substance, the effect of the test substance as a prophylactic or therapeutic agent is determined using the bone morphology of the lesion as an index Alternatively, a screening method for therapeutic agents.
(12) After administering the test substance, the effect of the test substance as a prophylactic or therapeutic agent is determined using the expression of miRNA-340 as an index, and the spinal ligament ossification according to (10) or (11) Screening method for preventive or therapeutic agent for symptom.
(13) A method for detecting ligamentous ossification lesion cells of the spinal ligament, comprising detecting miRNA-340 in a biological sample.
(14) The method for detecting vertebral ligament ossification lesion cells according to (13), wherein a probe capable of hybridizing with miRNA-340 is used.
(15) A kit for diagnosing ossification of the spinal ligament, comprising a probe capable of hybridizing with miRNA-340.
(16) A therapeutic agent for spinal ligament ossification, comprising as an active ingredient a nucleic acid capable of hybridizing with miRNA-340 or an expression vector capable of expressing the nucleic acid in the administration subject.

According to the spinal ligament ossification model non-human animal of the present invention and the spinal ligament ossification model cell of the present invention, a model of spinal ligament ossification can be provided. Further, according to the non-human animal of the spinal ligament ossification model of the present invention and the spinal ligament ossification model cell of the present invention, screening for a preventive or therapeutic agent for spinal ligament ossification can be performed using these. Can do. In addition, according to the spinal ligament ossification model non-human animal of the present invention and the spinal ligament ossification model cell of the present invention, it contributes to the elucidation of the onset mechanism of ossification of the spinal ligament.
The diabetes model non-human animal of the present invention and the diabetes model cell of the present invention can provide a model of diabetes.
According to the spinal ligament ossification disease cell detection method and spinal ligament ossification diagnosis kit of the present invention, it is possible to obtain information on the pathology of spinal ligament ossification.
According to the spinal ligament ossification treatment agent of the present invention, it becomes possible to treat spinal ligament ossification.

It is a schematic diagram showing the mouse genome replaced with a targeting vector and a targeting vector. It is the graph which compared the weight change of a knockout mouse (KO) and a wild type mouse (WT). It is the graph which compared the value of blood glucose and urine of a knockout mouse | mouth and a wild type mouse | mouth. It is the graph which compared the amount of drinking water of a knockout mouse and a wild type mouse. It is 3D-CT image of the longitudinal cross-section of the spine of a knockout mouse. It is a schematic diagram which shows the classification of posterior longitudinal ligament ossification. It is a photograph which shows the mode of ligament ossification seen in the posterior longitudinal ligament of a knockout mouse. It is a figure explaining the schedule of labeling substance administration for bone form measurement. It is a photograph which shows the mode of secretion of glucagon in the pancreatic islets of knockout mice. It is a HE-stained image of pancreatic tissue of a knockout mouse. It is the graph which compared the expression level of miR-340 in the plasma of a posterior longitudinal ligament ossification disease patient and a healthy person. It is an image which shows the analysis result of the mode of expression of miR-340 by in situ hybridization. It is the graph which compared the expression level of CXCL7 protein in the patient of the continuation type (continuation type) of posterior longitudinal ligament and the mixed type (patient). It is a graph which shows the result of having measured the cell which expresses BMP-2 protein which shows ossification by performing FACS (fluorescence activated cell sorting). (B) Measurement results for cells of ligament tissue prepared by introducing PRELP gene into equine mesenchymal stem cells, (A) Measurement results for cells of ligament tissue after knocking down the ligament tissue with shRNA-CXCL7. It is the dyeing | staining result which shows the grade of ossification in the cell by which CXCL7 was suppressed by shRNA, and the cell by which miR-340 inhibitor was suppressed by miR-340 inhibitor. It is the graph which compared the expression level of CXCL7 and the ossification marker in the cell by which CXCL7 was suppressed by shRNA, the cell by which miRNA-340 was introduce | transduced, and the cell by which miR-340 was suppressed by miR-340 inhibitor. It is an image which shows the electrophoretic image of TRAF3 and ubiquitin extracted from the cell of the spinal tissue. It is an image which shows the result of having performed ubiquitin antibody dyeing | staining with respect to the spinal tissue of a knockout mouse, and the spinal tissue of a human OPLL patient.

≪Non-human animal model of ossification of spinal ligament≫
The spinal ligament ossification model non-human animal of the present invention has CXCL7 gene function deficient or suppressed, or miRNA-340 expression is enhanced.

<First embodiment>
The spinal ligament ossification model non-human animal according to the present embodiment is one in which the function of the CXCL7 gene is deficient or suppressed.
A gene function deficient includes both a state in which the gene product is not present in the animal body or a state in which the function is lost even if the gene product is present in the animal body. That the function of CXCL7 gene is deficient or suppressed refers to a state in which the function of CXCL7 is significantly deficient or suppressed as compared to the normal group (wild type) of model animal species. Suppressing gene function means that the gene function is suppressed to such an extent that a pathological condition or trait characteristic of spinal ligament ossification appears or that ossification of spinal ligament can occur. Point to. Examples of gene products include mRNA, which is a transcription product, and proteins in which it is translated. It is known that deficiency and suppression of gene function in an animal body are caused by a change in the structure of the gene product, a decrease in the amount of the gene product, and the like.
Therefore, the defect or suppression of the function of the CXCL7 gene can be exemplified by the deletion, insertion, addition, or substitution of part or all of the CXCL7 gene or its expression control region.

  The CXCL7 gene is a CXCL7 structural gene, and examples of the expression control region of the CXCL7 gene include promoters, silencers, enhancers and the like.

  The “non-human animal” is not particularly limited as long as it is an animal other than a human, but is preferably a mammal, and more preferably an animal classified as a rodent. Non-human animals include mice, rats, guinea pigs, hamsters, rabbits, goats, pigs, dogs, cats, monkeys.

The amino acid sequence represented by SEQ ID NO: 1 is the amino acid sequence of human CXCL7 protein. The base sequence represented by SEQ ID NO: 2 is the base sequence (coding region sequence) of the human CXCL7 gene.
The amino acid sequence represented by SEQ ID NO: 3 is the amino acid sequence of a mouse (Mus musculus) CXCL7 protein (Ppbp (pro-platelet basic protein) protein). The base sequence represented by SEQ ID NO: 4 is the base sequence (coding region sequence) of the mouse CXCL7 gene (ppbp gene).

  The CXCL7 gene of a non-human animal to be a model animal can be determined based on the information of those already reported to be orthologs of human or mouse CXCL7 gene. For those not reported to be orthologs of the human or mouse CXCL7 gene, those skilled in the art can use, for example, a known technique based on the sequence of the human or mouse CXCL7 gene. The homologue and orthologue of the human or mouse CXCL7 gene in each non-human animal can be determined.

As an example of the CXCL7 gene of a non-human animal to be a model animal, a polynucleotide encoding any of the following polypeptides (A) to (C) can be mentioned.
(A) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 3,
(B) a polypeptide comprising an amino acid sequence in which one or several amino acids of the amino acid sequence represented by SEQ ID NO: 3 are deleted, substituted or added, and having an activity of improving ossification of the vertebral ligament of the spine
(C) a polypeptide comprising an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 3 and having an activity to improve ossification of the vertebral ligament

  In the polypeptide (B), the number of amino acids deleted, substituted or added to the amino acid sequence represented by SEQ ID NO: 3 is preferably 1-30, preferably 1-20, 10 is more preferable, and 1 to 5 is more preferable.

  In the polypeptide (C), the identity with the amino acid sequence represented by SEQ ID NO: 3 is not particularly limited as long as it is 80% or more, but it is preferably 85% or more, preferably 90% or more. Is more preferably 95% or more, and further preferably 98% or more.

  The identity between amino acid sequences can be determined using various homology search software known in the art. The identity value of amino acid sequences in the present invention can be obtained by calculation based on the alignment obtained by known homology search software BLAST.

  When a splicing variant or a truncated protein is present in the CXCL7 gene of a non-human animal serving as a model animal, these are also included as CXCL7 gene products.

  When the model animal of this embodiment has a plurality of CXCL7 alleles, deletion, insertion, addition, or substitution of the CXCL7 gene shows a pathological condition or trait characteristic of vertebral ligament ossification in the animal that becomes the model animal Alternatively, a part or all of deletions, insertions, additions, or substitutions in some CXCL7 alleles may be used as long as the animal to be a model animal develops ossification of the spinal ligament. Taking a mouse as an example, for example, the function of the CXCL7 gene may be deleted or suppressed by deletion, insertion, addition, or substitution in one CSCL7 gene of two CXCL7 alleles.

<Second embodiment>
The spinal ligament ossification model non-human animal of the present embodiment has miRNA-340 expression enhanced. In addition, description is abbreviate | omitted about the point which is common in [first embodiment] described previously.
The RNA sequences represented by SEQ ID NOs: 5 and 6 are the nucleic acid sequences of human and mouse miRNA-340, respectively.
The expression of miRNA being enhanced refers to a state where the expression level of miRNA is significantly increased as compared with the normal group (wild type) of model animal species.
The enhancement of miRNA expression can be caused, for example, by introducing miRNA-340 into the body of an animal that is a model animal. Here, miRNA-340 corresponds to miRNA precursor represented by Primary miRNA (pri-miRNA), miRNA-340 corresponding region and miRNA precursor encoded on the genome in addition to miRNA-340 itself. Includes area. The introduction of miRNA-340 may be introduced into the genome or may be introduced into the animal body and not introduced into the genome.

It is preferable that the non-human animal of the spinal ligament ossification disease model of the present invention has a pathological condition or trait characteristic to spinal ligament ossification disease. Spinal ligament ossification model non-human animal of the present invention is spinal ligament ossification, limb paralysis, low turnover bone condition, thinning of spinal cortical bone width, diabetes onset, type I diabetes onset, hyperglycemia, high glucagon value More preferably, it has at least one disease state or trait selected from the group consisting of diabetic nephropathy, high value indicating obesity of BMI (Body Math Index), and high expression of miRNA-340. These pathologies or traits are pathological conditions or traits that are characteristic of spinal ligament ossification.
Even in an individual derived from a non-human animal of the spinal ligament ossification model of the present invention (next-generation individual, etc.), the function of the CXCL7 gene is deficient or suppressed, or the expression of miRNA-340 is enhanced, And when it has a pathological condition or character characteristic of spinal ligament ossification, it is included in the non-human animal of the spinal ligament ossification disease model of the present invention.

Human spinal ligament ossification patients present with hyperglycemic conditions and may develop diabetes. In addition, CXCL7 knockout mice prepared in Examples described later also showed diabetes pathology as in humans. From this, the non-human animal of the spinal ligament ossification model of the present invention is a diabetic model non-human animal characterized in that the function of the CXCL7 gene is deficient or suppressed, or the expression of miRNA340 is enhanced. Include.
The diabetes model non-human animal of the present invention is from the group consisting of diabetes onset, type I diabetes onset, hyperglycemia, high glucagon level, diabetic nephropathy, and high value indicating obesity in BMI (Body Math Index). More preferably, it has at least one disease state or trait selected.

  The method for producing a spinal ligament ossification model non-human animal of the present invention and a diabetic model non-human animal of the present invention is a model non-human animal that lacks or suppresses the function of the CXCL7 gene or miRNA-340 The expression is not particularly limited as long as it can enhance the expression, and it is represented by partial or complete deletion, insertion, addition, or substitution in the CXCL7 gene or its expression control region, or introduction of miRNA-340, respectively. . These can be performed according to a known method for producing a transgenic animal or the like. For example, artificial mutation introduction into the genome, knockout or knockdown by gene targeting, knockdown using RNA interference, ribozyme, etc. can be exemplified.

As a method for inducing RNA interference targeting CXCL7, an RNAi-inducible nucleic acid capable of suppressing the expression of CXCL7 can be introduced into cells of a non-human animal serving as a model animal. Examples of RNAi-inducible nucleic acids include those designed based on the base sequence represented by SEQ ID NO: 4, and a sense strand consisting of all or a partial sequence of the base sequence represented by SEQ ID NO: 4, and complementary thereto And a nucleic acid having an antisense strand consisting of a simple sequence. Examples of the sequence length of the partial sequence include 19 to 341 bases, 20 to 100 bases, 21 to 39 bases, and the like.
The sense strand consisting of a partial sequence of the base sequence represented by SEQ ID NO: 4 is particularly preferably a base sequence composed of the 5th to 23rd bases in the base sequence represented by SEQ ID NO: 9.

  Deletion, insertion, addition, or substitution of part or all of the CXCL7 gene or its expression control region can also be carried out by methods common to those skilled in the art. Introduction of miRNA-340 into an animal to be a model animal can also be performed by methods common to those skilled in the art. For example, as shown in the below-mentioned Example, it can implement by the gene recombination technique using a Cre / loxP system.

As one embodiment of the present invention, the above-mentioned non-human animal in which the function of CXCL7 gene is deficient or suppressed or the expression of miRNA-340 is increased is used as a non-human animal with spinal ligament ossification disease model Provide a way to do it.
As one embodiment of the present invention, there is provided a method of using the above-mentioned non-human animal in which the function of CXCL7 gene is deficient or suppressed or in which miRNA-340 expression is enhanced as a diabetes model non-human animal. To do.

≪Spine ligament ossification model cell ・ ・ Diabetes model cell≫
The spinal ligament ossification model cell of the present invention is one in which the function of CXCL7 gene is deficient or suppressed, or the expression of miRNA-340 is enhanced.
The state in which the function of the CXCL7 gene is deficient or suppressed, or the expression of miRNA-340 is enhanced is the same as that described in the above-mentioned “vertebral ligament ossification model non-human animal”. In addition, regarding the spinal ligament ossification model cell, the spinal ligament ossification model cell production method, and the diabetes model cell, the content described in the above << Spine ligament ossification model non-human animal >> is replaced with the cell. Description of points that can be explained by this will be omitted.

The cell type of the vertebral ligament ossification model cell is not particularly limited, but it is preferably a parallel fibrous connective tissue cell, and more preferably a tendon / ligament cell. “Parallel fibrous connective tissue” refers to a close connective tissue in which collagen fibers are arranged in parallel in one direction, such as a tendon or a ligament. A cell of parallel fibrous connective tissue is a cell constituting a normal parallel fibrous connective tissue. The tendon or ligament cell is a cell constituting a normal tendon or ligament.
Parallel fibrous connective tissue cells obtained by differentiation induction from other types of cells or parallel fibrous connective tissues obtained by differentiation induction from other types of cells Other cells may be used. Tendon or ligament cells are tendon or ligament cells obtained by differentiation induction from other types of cells, or tendon-like or ligament-like cells obtained by differentiation induction from other types of cells. May be.
Examples of other types of cells include pluripotent cells, stem cells (artificial pluripotent stem cells, embryonic stem cells, mesenchymal stem cells), tendon / ligament progenitor cells, and fibroblasts. It is not restricted to cells.
A parallel fibrous connective tissue-like cell is one that has at least one of the various phenotypes prominent in known parallel fibrous connective tissue precursor cells or parallel fibrous connective tissue cells. A tendon-like or ligament-like cell is one having at least one of various phenotypes prominent in known tendon / ligament progenitor cells or tendon / ligament cells.

  The spinal ligament ossification model cell of the present invention can be used as a spinal ligament ossification model similar to the non-human animal of the spinal ligament ossification model of the present invention, and is easy to manufacture and handle, and is convenient. Excellent.

The method for producing the vertebral ligament ossification model cell can be carried out by deleting or suppressing the function of the CXCL7 gene in cells of parallel fibrous connective tissue or by enhancing the expression of miRNA-340. The method of deleting or suppressing the function of the CXCL7 gene in cells of parallel fibrous connective tissue or enhancing the expression of miRNA-340 is the same as that described in the above << Non-human animal with spinal ligament ossification disease model >>. Therefore, the description is omitted.
The method for producing a vertebral ligament ossification model cell can be carried out, for example, by suppressing the expression of CXCL7 by RNA interference with the CXCL7 gene, as shown in Examples described later. Examples of RNA interference include introduction of siRNA, shRNA, antisense nucleic acid and the like against CXCL7 into cells.
The cells of the parallel fibrous connective tissue may be induced to differentiate from other types of cells. Examples of the other types of cells include those described in the above-mentioned spinal ligament ossification model cells, and mesenchymal stem cells are preferable.

  As a method of obtaining differentiation from mesenchymal stem cells to obtain parallel fibrous connective tissue, Proline / arginine-rich end leucine- described in JP 2013-94098 A "Method for producing parallel fibrous connective tissue" A method using rich repeat protein (PRELP) can be mentioned.

  As a target for deleting or suppressing the function of the CXCL7 gene or enhancing the expression of miRNA-340, cells of parallel fibrous connective tissue or parallel fibrous connective tissue are preferable. In addition, since the parallel fibrous connective tissue may be obtained by differentiation induction from other types of cells, the function of the CXCL7 gene is lost or suppressed in any cell, or the expression of miRNA-340 is performed. May be induced to differentiate into parallel fibrous connective tissue or cells of parallel fibrous connective tissue.

Therefore, as one embodiment of the method for producing spinal ligament ossification model cells,
After culturing mesenchymal stem cells in contact with PRELP, a nucleic acid capable of deleting or suppressing the function of the CXCL7 gene by RNA interference with the cultured mesenchymal stem cells, or a vector capable of expressing the nucleic acid Can be exemplified by a method for producing a model cell for ossification of the vertebral column ligament including the introduction of
Moreover, as another embodiment of the method for producing another spinal ligament ossification model cell,
The mesenchymal stem cells are cultured in a state of contact with PRELP after introducing a nucleic acid capable of deficient or suppressing the function of the CXCL7 gene by RNA interference or a vector capable of expressing the nucleic acid into the mesenchymal stem cells. And a method for producing a vertebral ligament ossification model cell.

Furthermore, as one embodiment of a method for producing spinal ligament ossification model cells,
After culturing mesenchymal stem cells in contact with PRELP, a nucleic acid capable of hybridizing with miRNA-340 or the nucleic acid can be expressed in the administration subject to the cultured mesenchymal stem cells. An example is a method for producing a spinal ligament ossification model cell, which includes introducing an expression vector.
Moreover, as another embodiment of the method for producing another spinal ligament ossification model cell,
The mesenchymal stem cell is introduced into the mesenchymal stem cell after introducing a nucleic acid capable of hybridizing with miRNA-340, or an expression vector capable of expressing the nucleic acid in the administration subject, and then in contact with PRELP. Can be exemplified by a method for producing a model cell for ossification of the vertebral column ligament, which comprises culturing the vertebrae.

  As in the non-human animal of the spinal ligament ossification model of the present invention, the spinal ligament ossification model cell of the present invention has a CXCL7 gene function deficient or suppressed, or miRNA340 expression is enhanced. Includes diabetes model cells.

As one embodiment of the present invention, there is provided a method of using the above-mentioned cell in which the function of CXCL7 gene is deficient or suppressed, or the expression of miRNA-340 is enhanced, as a vertebral ligament ossification model cell To do.
As one embodiment of the present invention, there is provided a method of using the above-mentioned cell in which the function of CXCL7 gene is deficient or suppressed or the expression of miRNA-340 is enhanced as a diabetes model cell.

≪Screening method≫
The method of screening for a prophylactic or therapeutic agent for spinal ligament ossification according to the present invention includes a method for screening a ligament ossification model non-human animal of the present invention, a cell obtained from the spinal ligament ossification model non-human animal, The test substance is administered to the spinal ligament ossification model cell of the present invention. The test substance to be administered is a candidate for an active ingredient of a prophylactic or therapeutic agent for spinal ligament ossification.
There is no restriction | limiting in particular as a test substance, For example, a peptide, protein, a non-peptidic compound, a low molecular weight compound, a synthetic compound, a fermentation product, a cell extract, a plant extract, an animal tissue extract etc. are mentioned.
Spinal ligament ossification model cell of the present invention, spinal ligament ossification model non-human animal according to the present invention, and tissues and organs that are aggregates of the cells are also spinal ligament ossification model non-human animal Therefore, it can be used in a screening method for a prophylactic or therapeutic agent for spinal ligament ossification. In addition, when an individual obtained by crossing with a non-human animal of the vertebral ligament ossification model also has a pathological condition or trait characteristic of vertebral ligament ossification, a preventive or therapeutic agent for ossification of the vertebral ligament It can be used for screening methods.

  Since the spinal ligament ossification model non-human animal of the present invention and the vertebral ligament ossification model cell of the present invention exhibit pathological conditions or traits characteristic of spinal ligament ossification, the test substance is administered / not administered. The test substance can be screened for a prophylactic or therapeutic agent for spinal ligament ossification using the difference in these pathological conditions or traits as an index. The trait may be any item that can be measured, such as spinal ligament ossification, bone morphology, blood component, behavioral pattern, gene expression level, and expression level of bone tissue markers such as BMP-2 and RANKL. In particular, when using a non-human animal of a vertebral ligament ossification model, it is preferable to see the ossification of the spinal ligament, which is the most characteristic feature of the vertebral ligament ossification, in order to accurately grasp the effect of the test substance. When using a spinal ligament ossification model cell, it is preferable to measure the amount of marker protein in bone tissue in order to accurately grasp the effect of the test substance.

As one embodiment of the screening method for a prophylactic or therapeutic agent for spinal ligament ossification of the present invention, after administering the test substance, the bone morphology of the lesion is used as an index as a prophylactic or therapeutic agent for the test substance. It is mentioned to judge the effect.
For example, in the case where the expression of a bone tissue marker is used as an index, the bone tissue marker in the group in which the test substance is administered to the spinal ligament ossification model cell of the present invention is compared with the group in which the test substance is not administered. When the expression of is decreased, it can be determined that the administered test substance can be a prophylactic or therapeutic agent for spinal ligament ossification.
In ossification of the spinal ligament, it shows a characteristic pathological condition that a portion that should originally be a ligament ossifies. Therefore, whether or not the test substance can be a preventive or therapeutic agent for spinal ligament ossification, using as an index the improvement in bone morphology abnormally formed in the ligament after administration of the test substance Whether it can be determined. Examples of bone morphology measurement items include bone mass, bone area, number of osteoblasts, calcification rate, and osteoclast surface. For example, when the change in bone mass is used as an index, the group in which the test substance is administered to the non-human animal of the spinal ligament ossification model of the present invention is different from the group in which the test substance is not administered in comparison with the group to which the test substance is not administered. When the bone mass of the formed bone is decreased, it can be determined that the administered test substance can be a prophylactic or therapeutic agent for ossification of the spinal ligament.

  In measuring the bone form, the bone may be labeled using a labeling substance. By labeling the bone, information on the bone morphology can be obtained more clearly. Examples of the labeling substance include antibodies, fluorescent substances, alizarin and the like. Among them, it is preferable to use a fluorescent calcium chelating agent such as tetracycline or calcein as a labeling substance. By administering the fluorescent calcium chelating agent at the same time as the test substance, bones formed during the test substance administration period can be labeled.

As an embodiment of the screening method for a prophylactic or therapeutic agent for spinal ligament ossification according to the present invention, after administering the test substance, the expression of miRNA-340 is used as an index as a prophylactic or therapeutic agent for the test substance. It is mentioned to judge the effect.
As shown in Examples described later, the present inventors have found that the expression level of miRNA-340 is increased in the blood of human posterior longitudinal ligament ossification patients. Therefore, when the expression level of miRNA-340 is lower in the group in which the test substance is administered to the non-human animal of the spinal ligament ossification model according to the present invention, compared to the group in which the test substance is not administered, the administered test It can be determined that the substance can be an active ingredient of a prophylactic or therapeutic agent for spinal ligament ossification.
Since the measurement of the expression level of miRNA-340 can be easily performed by a conventionally known method such as PCR, screening can be performed more effectively by using the expression level of miRNA-340 as an index.

As one embodiment of the screening method for a prophylactic or therapeutic agent for spinal ligament ossification according to the present invention, after administering the test substance, phosphorylation of E3 ubiquitin ligase is used as an index as a prophylactic or therapeutic agent for the test substance. It is mentioned to judge the effect of.
As shown in Examples described later, the present inventor detected phosphorylation of E3 ubiquitin ligase in cells of spinal tissue of CXCL7 knockout mice. Therefore, in the group in which the test substance is administered to the non-human animal of the spinal ligament ossification model of the present invention, the phosphorylation level of E3 ubiquitin ligase is low or absent compared to the group to which the test substance is not administered. Thus, it can be determined that the administered test substance can be an active ingredient of a prophylactic or therapeutic agent for spinal ligament ossification. Detection of phosphorylation of E3 ubiquitin ligase can be performed by a known method such as mass spectrometry.
The amino acid sequence of mouse E3 ubiquitin ligase is shown in SEQ ID NO: 12. As shown in the examples described later, in the spinal tissue cells of CXCL7 knockout mice, at least the 419th serine and the 427th serine in the amino acid sequence represented by SEQ ID NO: 12 are phosphorylated. found. Therefore, phosphorylation of E3 ubiquitin ligase may be detected by detecting phosphorylation of these amino acids.
Even if it is E3 ubiquitin ligase other than SEQ ID NO: 12, E3 ubiquitin ligase is detected by detecting phosphorylation at the amino acid site corresponding to the 419th serine and / or the 427th serine in the amino acid sequence shown in SEQ ID NO: 12. May be detected.

As one embodiment of the screening method for a prophylactic or therapeutic agent for spinal ligament ossification according to the present invention, the test substance is administered using TRAF3 (tumor necrosis factor-associated facor 3) phosphorylation as an index after the test substance is administered. And determining the effect as a prophylactic or therapeutic agent.
As shown in Examples described later, the present inventor detected phosphorylation of TRAF3 in cells of the spinal tissue of CXCL7 knockout mice. Therefore, in the group in which the test substance is administered to the non-human animal of the spinal ligament ossification model of the present invention, when the level of phosphorylation of TRAF3 is low or absent compared to the group to which the test substance is not administered, It can be determined that the test substance can be an active ingredient of a prophylactic or therapeutic agent for spinal ligament ossification. The measurement of phosphorylation of TRAF3 can be performed by a known method such as mass spectrometry.
The amino acid sequence of mouse TRAF3 is shown in SEQ ID NO: 13. As shown in Examples described later, at least the 211th threonine of the amino acid sequence represented by SEQ ID NO: 13, the 412th serine, and the 413th tyrosine are phosphorylated in cells of the spinal tissue of CXCL7 knockout mice. Turned out to be. Therefore, TRAF3 phosphorylation may be detected by detecting phosphorylation of these amino acids.
Even for TRAF3 other than SEQ ID NO: 13, any one or more of phosphorylation at the amino acid site corresponding to the 211th threonine, 412th serine, and 413th tyrosine of the amino acid sequence represented by SEQ ID NO: 13 By detecting, phosphorylation of TRAF3 may be detected.

As one embodiment of the screening method for a prophylactic or therapeutic agent for ossification of the spinal ligament of the present invention, after the administration of the test substance, the test is performed using phosphorylation of TZF-L (zinc finger protein TZF-L) as an index. It is mentioned to judge the effect of a substance as a prophylactic or therapeutic agent.
As shown in Examples described later, the present inventor detected phosphorylation of TZF-L in the spinal tissue cells of CXCL7 knockout mice. Therefore, in the case where the test substance is administered to the non-human animal of the spinal ligament ossification model of the present invention, the level of phosphorylation of TZF-L is lower or absent compared to the group not administered with the test substance Thus, it can be determined that the administered test substance can be an active ingredient of a prophylactic or therapeutic agent for spinal ligament ossification. The measurement of phosphorylation of TZF-L can be performed by a known method such as mass spectrometry.
The amino acid sequence of mouse TZF-L is shown in SEQ ID NO: 15. As shown in Examples described later, in cells of the spinal tissue of CXCL7 knockout mice, at least the 677th serine, the 1652th methionine, and the 1668th serine of the amino acid sequence represented by SEQ ID NO: 15 are phosphorylated. Turned out to be. Therefore, phosphorylation of TZF-L may be detected by detecting phosphorylation of these amino acids.
Even if TZF-L is other than SEQ ID NO: 15, any one of phosphorylation at the amino acid site corresponding to the 677th serine, the 1652th methionine, and the 1668th serine of the amino acid sequence represented by SEQ ID NO: 15 By detecting the above, phosphorylation of TZF-L may be detected.

  Each index which judges the effect as a preventive agent or therapeutic agent of the test substance demonstrated above may be used independently, respectively, and may be used in combination.

As one embodiment of the present invention, a screening method for a prophylactic or therapeutic agent for diabetes is provided. A screening method for a prophylactic or therapeutic agent for diabetes is to administer a test substance to a non-human animal of the diabetes model of the present invention, to a cell obtained from the non-human animal of the diabetes model, or to a diabetes model cell of the present invention. .
There is no restriction | limiting in particular as a test substance, For example, a peptide, protein, a non-peptidic compound, a low molecular weight compound, a synthetic compound, a fermentation product, a cell extract, a plant extract, an animal tissue extract etc. are mentioned.
Since the diabetes model cell of the present invention, the cell obtained from the diabetes model non-human animal according to the present invention, and the tissues and organs that are aggregates of the cells also have a phenotype derived from the diabetes model non-human animal, It can be used in a screening method for a prophylactic or therapeutic agent for diabetes. In addition, when an individual obtained by crossing with a diabetes model non-human animal also has a disease state or trait characteristic of diabetes, it can be used in a screening method for a prophylactic or therapeutic agent for diabetes.

  The non-diabetic model human animal of the present invention and the diabetic model cell of the present invention exhibit a pathological condition or trait characteristic of diabetes. Substances can be screened for preventive or therapeutic agents for diabetes. The trait may be any item that can be measured, such as diabetes onset, type I diabetes onset, hyperglycemia, high glucagon levels, blood components, behavioral patterns, gene expression levels, and expression levels of diabetes markers. In particular, when using a non-diabetic model animal, it is preferable to observe hyperglycemia, which is the most characteristic of diabetes, in order to accurately grasp the effect of the test substance. When using a diabetes model cell, it is preferable to measure the amount of the marker protein for diabetes in order to accurately grasp the effect of the test substance.

As one embodiment of a method for screening a prophylactic or therapeutic agent for diabetes according to the present invention, after the administration of the test substance, the preventive or treatment of the test substance using as an index a value indicating the presence of blood components related to diabetes It is mentioned to judge the effect as an agent.
For example, when the blood glucose level is used as an indicator, the group administered with the test substance to the diabetes model cell of the present invention is compared with the group not administered with the test substance. It can be determined that the substance can be a prophylactic or therapeutic agent for diabetes.

As one embodiment of the screening method for a prophylactic or therapeutic agent for diabetes according to the present invention, after administering the test substance, the effect of the test substance as a prophylactic or therapeutic agent is determined using phosphorylation of adiponectin as an index. Is mentioned.
As shown in Examples described later, the present inventor detected adiponectin phosphorylation in the blood of CXCL7 knockout mice. Therefore, in the group in which the test substance is administered to the non-human animal model of diabetes of the present invention, when the level of phosphorylation of adiponectin is low or absent compared to the group not administered with the test substance, the administered test substance is It can be determined that it can be an active ingredient of a prophylactic or therapeutic agent for diabetes. Adiponectin phosphorylation can be measured by a known method such as mass spectrometry.
The amino acid sequence of mouse adiponectin is shown in SEQ ID NO: 14. As shown in Examples described later, at least the 64th threonine, the 119th serine and the 127th threonine of the amino acid sequence represented by SEQ ID NO: 14 are phosphorylated in cells of the spinal tissue of CXCL7 knockout mice. Turned out to be. Therefore, phosphorylation of TZF-L may be detected by detecting phosphorylation of these amino acids.
Even for adiponectin other than SEQ ID NO: 14, any one or more of phosphorylation at the amino acid site corresponding to the 64th threonine, the 119th serine and the 127th threonine of the amino acid sequence represented by SEQ ID NO: 14 is detected By doing so, phosphorylation of adiponectin may be detected.

  Each index which judges the effect as a preventive agent or therapeutic agent of the test substance demonstrated above may be used independently, respectively, and may be used in combination.

≪Method for detecting vertebral ligament ossification lesion cells≫
The method for detecting a vertebral ligament ossification lesion cell of the present invention detects miRNA-340 in a biological sample. The biological sample includes a sample collected from a patient suspected of having vertebral ligament ossification, and includes a sample used for biopsy.
The present inventor has found that the expression level of miRNA-340 in a biological sample derived from a patient with ossification of the spinal ligament has increased. Therefore, by detecting miRNA-340 in a biological sample, vertebral ligament ossification lesion cells can be detected. In addition, when the expression level of miRNA-340 in the biological sample derived from the subject is significantly increased compared to the expression level of miRNA-340 in the biological sample derived from a healthy person who is a normal control group, It has vertebral ligament ossification lesion cells and can detect the affliction of spinal ligament ossification.

A probe capable of hybridizing with miRNA-340 may be used in the method of detecting vertebral ligament ossification lesion cells of the present invention. Here, miRNA-340 may be a miRNA precursor represented by Primary miRNA (pri-miRNA) in addition to miRNA-340, and is preferably miRNA-340.
An example of a probe that can hybridize with miRNA-340 is a polynucleotide having a base sequence complementary to the base sequence represented by SEQ ID NO: 5. In addition, in the base sequence represented by SEQ ID NO: 5, one to several bases are deleted, substituted, or added as long as it can specifically hybridize with miRNA-340 Even a polynucleotide having a can be used similarly as a probe. Here, 1 to several is preferably 1 to 5, and more preferably 1 to 3. As long as the probe can hybridize with miRNA-340, any substance such as a labeling substance or an enzyme is added, or various chemical modifications are included, and at least a part thereof is PNA, LNA, etc. Or a nucleic acid analog of

  The present inventor detected phosphorylation of E3 ubiquitin ligase in cells of spinal tissue of CXCL7 knockout mice. Therefore, by detecting phosphorylation of E3 ubiquitin ligase in a biological sample, vertebral ligament ossification lesion cells can be detected. In addition, the phosphorylation level of E3 ubiquitin ligase in a biological sample derived from a subject is detected or the phosphorylation level is compared with the phosphorylation level of E3 ubiquitin ligase in a biological sample derived from a healthy person who is a normal control group. If significantly increased, the subject has spinal ligament ossification lesion cells and can detect the affliction of spinal ligament ossification.

By detecting phosphorylation of E3 ubiquitin ligase in a biological sample and phosphorylation of 419th serine and / or 427th serine in the amino acid sequence shown in SEQ ID NO: 12, vertebral ligament ossification lesion cell Can be detected.
The E3 ubiquitin ligase in which the serine at 419 and / or the 427th serine in the amino acid sequence shown in SEQ ID NO: 12 is phosphorylated can be used as a marker for spinal ligament ossification lesion cells.
A polypeptide comprising an E3 ubiquitin ligase fragment polypeptide comprising a phosphorylation of serine 419 and / or 427 in the amino acid sequence shown in SEQ ID NO: 12 is used as a marker for spinal ligament ossification lesion cells Is possible.

Even an E3 ubiquitin ligase other than SEQ ID NO: 12 is phosphorylation of E3 ubiquitin ligase in a biological sample and corresponds to the 419th serine and / or the 427th serine in the amino acid sequence shown in SEQ ID NO: 12. By detecting phosphorylation at the amino acid site, vertebral ligament ossification lesion cells can be detected.
Even if it is an E3 ubiquitin ligase other than SEQ ID NO: 12, the E3 ubiquitin ligase in which the amino acid site corresponding to the 419th serine and / or the 427th serine in the amino acid sequence shown in SEQ ID NO: 12 is phosphorylated is a spinal ligament It can be used as a marker for ossification lesion cells.
Even if it is an E3 ubiquitin ligase other than SEQ ID NO: 12, it is an E3 ubiquitin ligase fragment polypeptide, which is a 419th serine and / or 427th serine in the amino acid sequence shown in SEQ ID NO: 12. Polypeptides containing oxidation can be used as markers for vertebral ligament ossification lesion cells.

  The present inventor detected phosphorylation of TRAF3 in cells of spinal tissue of CXCL7 knockout mice. Therefore, by detecting phosphorylation of TRAF3 in a biological sample, vertebral ligament ossification lesion cells can be detected. In addition, when the phosphorylation level of TRAF3 in a biological sample derived from a subject is detected or compared with the phosphorylation level of TRAF3 in a biological sample derived from a healthy person who is a normal control group, the phosphorylation level is significantly increased. If so, the subject has vertebral ligament ossification lesion cells and can detect the affliction of vertebral ligament ossification.

Detecting at least one of phosphorylation of TRAF3 in a biological sample and phosphorylation of 211th threonine, 412th serine and 413th tyrosine in the amino acid sequence shown in SEQ ID NO: 13 Ligament ossification lesion cells can be detected.
TRAF3 phosphorylated at any one or more of the 211th threonine, the 412th serine, and the 413th tyrosine in the amino acid sequence shown in SEQ ID NO: 13 can be used as a marker for vertebral ligament ossification lesion cells. is there.
A polypeptide of TRAF3 fragment, comprising a phosphorylation of an amino acid site corresponding to any one or more of the 211th threonine, 412th serine, and 413th tyrosine in the amino acid sequence shown in SEQ ID NO: 13 The peptide can be used as a marker for spinal ligament ossification lesion cells.

Even TRAF3 other than SEQ ID NO: 13 is phosphorylation of TRAF3 in a biological sample, and any one of 211th threonine, 412th serine and 413th tyrosine in the amino acid sequence shown in SEQ ID NO: 13 By detecting phosphorylation of the amino acid site corresponding to the above, vertebral ligament ossification lesion cells can be detected.
Even in TRAF3 other than SEQ ID NO: 13, the amino acid site corresponding to one or more of the 211th threonine, 412th serine, and 413th tyrosine in the amino acid sequence shown in SEQ ID NO: 13 was phosphorylated. TRAF3 can be used as a marker for spinal ligament ossification lesion cells.
TRAF3 other than SEQ ID NO: 13 is a TRAF3 fragment polypeptide, and corresponds to any one or more of the 211th threonine, 412th serine, and 413th tyrosine in the amino acid sequence shown in SEQ ID NO: 13 Polypeptides containing phosphorylated amino acid sites that can be used as markers for vertebral ligament ossification lesion cells.

  The present inventor detected phosphorylation of TZF-L in cells of spinal tissue of CXCL7 knockout mice. Therefore, vertebral ligament ossification lesion cells can be detected by detecting phosphorylation of TZF-L in a biological sample. In addition, when the phosphorylation level of TZF-L in a biological sample derived from a subject is detected or the phosphorylation level is compared with the phosphorylation level of TZF-L in a biological sample derived from a healthy person who is a normal control group If significantly increased, the subject has spinal ligament ossification lesion cells and can detect the affliction of spinal ligament ossification.

Detecting at least one of phosphorylation of TZF-L in a biological sample and phosphorylation of 677th serine, 1652th methionine, and 1668th serine in the amino acid sequence shown in SEQ ID NO: 15 By this, vertebral ligament ossification lesion cells can be detected.
TZF-L phosphorylated at one or more of the 677th serine, the 1652th methionine, and the 1668th serine in the amino acid sequence shown in SEQ ID NO: 15 is used as a marker for vertebral ligament ossification lesion cells Is possible.
A polypeptide of a TZF-L fragment, which comprises phosphorylation of an amino acid site corresponding to any one or more of 677th serine, 1652th methionine, and 1668th serine in the amino acid sequence shown in SEQ ID NO: 15 The polypeptide can be used as a marker for spinal ligament ossification lesion cells.

Even TZF-L other than SEQ ID NO: 15 is a phosphorylation of TZF-L in a biological sample, and the 677th serine, 1652th methionine, and 1668th amino acid in the amino acid sequence shown in SEQ ID NO: 15 By detecting phosphorylation of amino acid sites corresponding to any one or more of serine, vertebral ligament ossification lesion cells can be detected.
Even in the case of TZF-L other than SEQ ID NO: 15, the amino acid site corresponding to one or more of the 677th serine, the 1652th methionine, and the 1668th serine in the amino acid sequence shown in SEQ ID NO: 15 is phosphorylated. TZF-L can be used as a marker for vertebral ligament ossification lesion cells.
Even if it is TZF-L other than SEQ ID NO: 15, it is a TZF-L fragment polypeptide, and any one of 677th serine, 1652th methionine, and 1668th serine in the amino acid sequence shown in SEQ ID NO: 15 Polypeptides comprising phosphorylation of amino acid sites corresponding to one or more can be used as markers for spinal ligament ossification lesion cells.

  The expression level of miRNA-340, phosphorylation of E3 ubiquitin ligase, phosphorylation of TRAF3, and phosphorylation of TZF-L described above may be used alone in detecting vertebral ligament ossification lesion cells, respectively. A plurality may be used in combination. When using in combination, a plurality of known factors used for detection of vertebral ligament ossification lesion cells may be used.

Explained spinal ligament ossification model non-human animal, spinal ligament ossification model cell, screening method for prophylactic or therapeutic agent for spinal ligament ossification, and detection method of spinal ligament ossification lesion cell Since ossification is associated with diabetes, ossification of the spinal ligament may be detected indirectly based on the traits used to represent diabetes, pathological conditions, indices, characteristics, and the like.
Similarly, a diabetes model non-human animal, a diabetes model cell, a screening method for a prophylactic or therapeutic agent for diabetes, spinal ligament ossification is associated with diabetes, and traits used to represent spinal ligament ossification, Diabetes may be detected indirectly based on the disease state, index, characteristics, and the like.

≪Diagnostic kit for ossification of spinal ligament≫
The kit for diagnosing spinal ligament ossification according to the present invention includes a probe capable of hybridizing with miRNA-340. Examples of the probe include the same probes as those described in << Method for detecting ligamentous ossification disease cell of spinal ligament >>.
In addition, from the viewpoint of simplifying the diagnosis of spinal ligament ossification, the kit for diagnosing spinal ligament ossification according to the present invention may be provided with reagents and instruments necessary for detection of miRNA-340. preferable. In this way, by making reagents, instruments, and the like necessary for detection of miRNA-340 into a kit, vertebral ligament ossification can be diagnosed more easily.

≪Spine ligament ossification treatment≫
One embodiment of the therapeutic agent for ossification of the spinal ligament of the present invention comprises a nucleic acid capable of hybridizing with miRNA-340 or an expression vector capable of expressing the nucleic acid in the administration subject as an active ingredient.
It is known that the function of miRNA is inhibited by hybridization of nucleic acid with miRNA. Therefore, since a nucleic acid capable of hybridizing with miRNA-340 inhibits the function of miRNA-340, it can be used as a therapeutic agent for spinal ligament ossification. Those in which a nucleic acid capable of hybridizing with miRNA-340 is incorporated into an arbitrary expression vector can also be used as a therapeutic agent for ossification of the spinal ligament. By using a vector, a long-term effect of treating ossification of the spinal ligament can be obtained.

  Similarly, a nucleic acid that can suppress the expression of miRNA-340, or an expression vector that can express the nucleic acid in the administration target as an active ingredient, and a function of miRNA-340 in competition with miRNA-340 Nucleic acids that can be inhibited, or those that contain an expression vector capable of expressing the nucleic acid in the administration subject as an active ingredient can also be exemplified as therapeutic agents for ossification of the spinal ligament.

<Pharmaceutical formulation>
Since the active ingredient of the medicament of the present invention is a nucleic acid that can hybridize with miRNA-340, or an expression vector that can express the nucleic acid in the administration subject, the dosage form is preferably a parenteral administration agent that is easily absorbed, An injection is more preferable. Examples of parenteral agents include injections, inhalants, patches, suppositories and the like.

(Injection)
Examples of the injection include intra-pathological ligament injection, intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection, infusion injection and the like. An injection is prepared by dissolving a nucleic acid capable of hybridizing with miRNA-340 or an expression vector capable of expressing the nucleic acid in a subject to be administered in a sterile aqueous or oily liquid (carrier for injection) usually used for injection, It can be prepared by suspending or emulsifying. Examples of the aqueous liquid for injection include isotonic solutions containing physiological saline, glucose and other adjuvants. Suitable solubilizers such as alcohol (eg, ethanol), polyalcohol (eg, propylene glycol, polyethylene glycol), nonionic surfactant [eg, polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)] and the like. Examples of the oily liquid include sesame oil and soybean oil. As a solubilizer, benzyl benzoate, benzyl alcohol, or the like may be used in combination.

The content of the nucleic acid capable of hybridizing with miRNA-340 in the injection may be, for example, about 0.01 to 100 w / v%, preferably about 0.1 to 30 w / v% in terms of dry weight.
The content of the expression vector capable of expressing in the administration subject a nucleic acid capable of hybridizing with miRNA-340 in the injection is, for example, about 0.01 to 100 w / v%, preferably about 0.1, in terms of dry weight. It should be ~ 30w / v%.

(Patch)
As a patch, a plaster, a poultice, a tape, a plaster in which a base comprising a nucleic acid capable of hybridizing with miRNA-340 or an expression vector capable of expressing the nucleic acid in a subject to be administered is supported. Agents and the like. Bases include sodium alginate, gelatin, corn starch, tragacanth gum, methyl cellulose, hydroxyethyl cellulose, carboxymethyl cellulose, xanthan gum, carrageenan, mannan, agarose, dextrin, carboxymethyl starch, polyvinyl alcohol, sodium polyacrylate, methoxyethylene-maleic anhydride Polymers such as copolymer, polyvinyl ether, polyvinyl pyrrolidone, carboxyvinyl polymer, hydroxypropylcellulose, hydroxypropylmethylcellulose, pullulan; hydrocarbons such as white petrolatum, yellow petrolatum, paraffin, ceresin wax, microcrystalline wax; gelation Hydrocarbons (for example, the trade name Plastibase, Bristol-Myers Squii Manufactured by the company); higher fatty acids such as stearic acid; higher alcohols such as cetanol, octyldodecanol and stearyl alcohol; polyethylene glycol (eg, Macrogol 4000); propylene glycol, glycerin, dipropylene glycol, 1,3-butylene glycol And polyhydric alcohols such as concentrated glycerin; fatty acid esters such as monooleic acid ester and stearic acid glyceride; and phosphate buffer. Furthermore, additives such as a solubilizing agent, an inorganic filler, a pH adjuster, a humectant, a preservative, a thickener, an antioxidant, and a cooling agent may be added.

The content of the nucleic acid capable of hybridizing with miRNA-340 in the base is, for example, about 0.01 to 100% by weight, preferably about 0.1 to 30% by weight in terms of dry weight.
The content of the expression vector capable of expressing in the administration subject a nucleic acid capable of hybridizing with miRNA-340 in the base is, for example, about 0.01 to 100% by weight, preferably about 0.1 to 30% in terms of dry weight. The weight% may be used.
These are used as attachment agents. However, for more effective implementation, it is recommended that ultrasonic attachment be performed after attachment. By ultrasonic penetration, the affected area can be more efficiently penetrated.

(Inhalant)
Examples of the inhalant include aerosols, inhalation powders, inhalation liquids (for example, inhalation solutions, inhalation suspensions, etc.), capsule inhalants and the like. The liquid for inhalation may be in a form to be used by dissolving or suspending in water or other suitable medium at the time of use.
Inhalants can be manufactured according to conventional methods. For example, a nucleic acid capable of hybridizing with miRNA-340, or an expression vector capable of expressing the nucleic acid in a subject to be administered is made into a dry powder or a liquid and mixed with an inhalation propellant, a carrier, or both, and appropriate inhalation Manufactured by filling containers. When a nucleic acid capable of hybridizing with miRNA-340 is pulverized, it can be finely powdered with lactose, starch, magnesium stearate and the like to form a uniform mixture or granulated to prepare a powder. In addition, when a nucleic acid that can hybridize with miRNA-340 is made liquid, it may be dissolved in a liquid carrier such as water, physiological saline, or buffer.
Examples of the propellant include conventionally known propellants such as alternative fluorocarbons, liquefied gas propellants (for example, fluorinated hydrocarbons, liquefied petroleum, diethyl ether, dimethyl ether, etc.), compressed gases (for example, soluble gases (for example, carbon dioxide) Nitrous oxide gas, etc.), insoluble gas (for example, nitrogen gas, etc.) and the like are used.

  The inhalant may further contain an additive as necessary. As an additive, in the case of an inhalation solution, an antiseptic (benzalkonium chloride, paraben, etc.), a coloring agent, a buffering agent (sodium phosphate, sodium acetate, etc.), an isotonic agent (for example, sodium chloride, Concentrated glycerin and the like), thickeners (for example, carboxyvinyl polymer and the like), absorption accelerators and the like. In the case of powders for inhalation, lubricants (eg, magnesium stearate, light anhydrous silicic acid, talc, sodium lauryl sulfate, etc.), flavoring agents (eg, citric acid, menthol, glycyrrhizin ammonium salt, glycine, orange) Powders, etc.), binders (eg starch, dextrin, methylcellulose, hydroxypropylcellulose, polyvinylpyrrolidone, polyethylene glycol, sucrose, etc.), excipients (eg sucrose, lactose, glucose, mannitol, sorbit, maltose, cellulose, etc.) ), Coloring agents, preservatives (eg, sodium benzoate, sodium bisulfite, methylparaben, propylparaben, etc.), stabilizers (eg, citric acid, sodium citrate, etc.), absorption enhancers (bile salts, chitosan, etc.) ) And the like.

The content of the nucleic acid capable of hybridizing with miRNA-340 in the spray is, for example, about 0.01 to 100 w / v%, preferably about 0.1 to 30 w / v in terms of dry weight in the case of a liquid for inhalation. %And it is sufficient. In the case of a powder for inhalation, it may be, for example, about 0.01 to 100% by weight, preferably about 0.1 to 30% by weight in terms of dry weight.
In the case of an inhalation solution, the content of the expression vector capable of expressing the nucleic acid capable of hybridizing with miRNA-340 in the propellant in the administration subject is, for example, about 0.01 to 100 w / in terms of dry weight. It may be v%, preferably about 0.1-30 w / v%. In the case of a powder for inhalation, it may be, for example, about 0.01 to 100% by weight, preferably about 0.1 to 30% by weight in terms of dry weight.

(Suppository)
Suppositories can be exemplified by those prepared by mixing a nucleic acid capable of hybridizing with miRNA-340 or an expression vector capable of expressing the nucleic acid in the administration subject with a commonly used suppository base.
The content of the nucleic acid capable of hybridizing with miRNA-340 in the suppository may be, for example, about 0.01 to 100% by weight, preferably about 0.1 to 30% by weight in terms of dry weight.
The content of the expression vector capable of expressing the nucleic acid capable of hybridizing with miRNA-340 in the suppository in the administration subject is, for example, about 0.01 to 100% by weight, preferably about 0.1 to 30% in terms of dry weight. The weight% may be used.

(Oral administration)
Examples of the orally administered agent include powders, granules, tablets, tablets, pills, capsules, chewable agents, emulsions, liquids, syrups and the like. Examples of solid preparations include those prepared by blending the above active ingredients with pharmaceutically acceptable carriers and additives. For example, excipients such as sucrose, lactose, glucose, starch, mannitol; binders such as gum arabic, gelatin, crystalline cellulose, hydroxypropylcellulose, methylcellulose; disintegrants such as carmellose, starch; anhydrous citric acid Stabilizers such as sodium laurate and glycerol are blended. Further, it may be coated or encapsulated with gelatin, white sugar, gum arabic, carnauba wax or the like. Examples of the liquid preparation include those prepared by dissolving or dispersing the above active ingredient in water, ethanol, glycerin, simple syrup, or a mixture thereof.

The content of the nucleic acid capable of hybridizing with miRNA-340 in the oral administration agent may be, for example, about 0.01 to 100% by weight, preferably about 0.1 to 50% by weight in terms of dry weight.
The content of the expression vector capable of expressing the nucleic acid capable of hybridizing with miRNA-340 in the administration subject in the oral administration agent is, for example, about 0.01 to 100% by weight, preferably about 0.1 to 100% by weight in terms of dry weight. It may be 30% by weight.

<How to use>
Examples of the administration route include injection administration (intra-pathological ligament injection, intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection, infusion), transdermal administration, intratracheal administration, rectal administration, oral administration, and the like. In particular, injection administration is preferable, and intra-pathological ligament injection is more preferable.
The therapeutic agent of the present invention is used for patients with ossification of the spinal ligament. Spinal ligament ossification includes posterior longitudinal ligament ossification, yellow ligament ossification, and anterior longitudinal ligament ossification. As a subject of the therapeutic agent of the present invention, posterior longitudinal ligament ossification patients and the like are particularly suitable.
The use amount of the spinal ligament ossification treatment agent of the present invention varies depending on the symptom and age of the subject, but in the case of parenteral administration, the daily dose for human adults is as follows.

That is, the daily dose of nucleic acid that can hybridize with miRNA-340 is preferably about 0.01 mg to 500 mg, more preferably about 0.1 mg to 200 mg, in terms of dry weight. In addition, it is preferable that the daily dose to humans of an expression vector capable of expressing a nucleic acid capable of hybridizing with miRNA-340 in a subject to be administered is about 0.1 μg to 1 g in terms of dry weight. More preferably, it is about 0.1 μg to 500 mg.
In the case of oral administration, the daily dose for human adults is as follows.
That is, the daily dose of nucleic acid that can hybridize with miRNA-340 is preferably about 0.1 mg to 1000 mg, more preferably about 1 mg to 500 mg, in terms of dry weight. In addition, the daily dose to humans of an expression vector capable of expressing a nucleic acid capable of hybridizing with miRNA-340 in a subject is preferably about 1 μg to 1000 mg in terms of dry weight, It is more preferable that it is 1 microgram-500 mg.

Administering an effective amount of a spinal ligament ossification agent to a human vertebral ligament ossification patient can be performed as a method for treating spinal ligament ossification. Examples of the effective amount of the spinal ligament ossification treatment agent include those exemplified above.
In the present invention, “treatment” includes healing, alleviating or ameliorating symptoms.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated more concretely, this invention is not limited to these.

[Preparation of CXCL7 knockout mouse]
Based on the nucleic acid sequence of the genomic sequence of the Ppbp gene (mouse CXCL7 gene), the inventors incorporated loxP sequences between exon 1 and exon 2 of the Ppbp genomic sequence and 3'arm side of exon 3 of the Ppbp genomic sequence. A targeting vector was constructed. FIG. 1 shows a schematic diagram of the constructed targeting vector and a schematic diagram of a genome to be replaced with the targeting vector. This targeting vector was introduced into C57BL / 6-derived RENKA, which is a mouse ES cell line, by electroporation. Among them, ES colony that caused homologous recombination was selected. Selected ES cells were transplanted into mouse blastocysts and introduced into germline cells. Next, a third generation (F3) mouse was prepared by natural mating of a second generation (F2) heterozygous mouse (PpbpdE ^{2E3 / +} : CAG-Cree) of a Ppbp gene conditional knockout mouse and a wild type mouse. Genomic DNA was collected from offspring tissues, and the genotype was analyzed by PCR analysis. The portion surrounded by the loxP sequence of the targeting vector was deleted with Cre, and exon 2 and exon 3 of the Ppbp gene were deleted to obtain a heterozygote (Ppbp ^{dE2E3 / +} ) of the Ppbp gene knockout mouse. The F3 mouse from which the Cre transgene was removed (Ppbp ^{dE2E3 / +} ) did not obtain the target two pairs, so the F3 heterozygous mouse from which the Cre transgene was removed (Ppbp ^{dE2E3 / +} ) and the wild type mouse F4 offspring were produced by natural mating.
After DNA extraction from the body tissue of F4 offspring, 9 F4 mice (3 ^♀, 6 ^♀ ) were heterozygous mice (Ppbp ^{dE2E3 / +} ) from which the Cre transgene was removed. It was determined that there was. And the homozygote (Ppbp ^{dE2E3 / dE2E3} ) of the conditional knockout mouse | mouth of a Ppbp gene was obtained by natural mating between heterozygotes. Hereinafter, a homozygote of a conditional knockout mouse for the Ppbp gene is abbreviated as “knockout mouse”.

  The knockout mouse homo male: female birth ratio was 2: 1. Since the male: female ratio of human vertebral ligament ossification patients is about 2: 1, this figure can be said to be similar to the male / female ratio of human vertebral ligament ossification patients.

[Phenotype of CXCL7 knockout mouse]
Until the second month after birth, there was no difference in body weight between wild-type mice and knockout mice. However, after 2 months of age, the body weight of the knockout mice gradually increased compared to the wild type mice (FIG. 2). In knockout mice, blood and urine glucose values and HbA1c levels were also increased compared to wild-type mice (FIG. 3). The knockout mouse was polydipuria compared with the wild type mouse (FIG. 4). As a result of measuring urinary potassium in knockout mice (homo), it was as high as 103.2 mEq / L (normal value 3.6-5.0 mEq / L). This value means insulin deficiency, renal failure, and the like. These results suggested that the mice developed diabetic nephropathy.
When the visceral fat of knockout mice was measured, the body mass index (BMI) was 50 or more. When the amounts of subcutaneous fat and visceral adipose tissue were confirmed by 3D micro X-ray CT (manufactured by Rigaku Corporation), the condition of subcutaneous fat and testis suggested fatty liver.
At 4 months of age, the motor function of the hind limbs of the knockout mice gradually decreased, making it difficult to walk. When mice are swimming in warm water, normally wild-type mice can swim by moving their hind limbs, but knockout mice show persistent stiffness in their hind limbs and significant functional impairment in their hind limbs. Was recognized.
Some knockout mice developed retinopathy.

FIG. 5 is a 3D-CT image of a knockout mouse (homo) 8 months old. The image was acquired with a longitudinal section of the spine. The ossification of the ligament was confirmed at the tip of the arrow in the figure. In the knockout mice, ossification of the spinal ligament occurred after 8 months (middle age) after birth.
The posterior longitudinal ligament ossification is classified into continuous type (A), segmental type (B), mixed type (C), and others: intervertebral disc limited type (D) according to the ossification state of the ligament (see FIG. 6). Journal of Bone and Mineral Metabolism, November 1999, Volume 17, Issue 4, pp 296-300). Of these types, the continuous type (A) is the most serious disease state. As a result of 3D-CT image analysis, the ossification state of the knockout mouse (homo) was continuous (A).

  FIG. 7 shows a photograph of the posterior longitudinal ligament of a knockout mouse (homo / male). The ligament ossification was clearly confirmed in the posterior longitudinal ligament of the first (L1) portion of the spine. As is clear from FIG. 7, a spherical bone tissue was observed, and a continuous bone tissue was also observed below it.

(Bone morphometry)
In order to easily perform bone morphometry, the bone morphometry was performed after the labeling substance was administered to the mouse to be measured.
FIG. 8 is a diagram for explaining a schedule of labeling substance administration to mice. Five days before Sacrifice, a labeling substance tetracycline (TC) was administered to mice as the first bone labeling treatment. 72 hours after the first bone labeling treatment, the labeling substance calcein (CL) was administered to the mice as the second bone labeling treatment. By these processes, bone metabolism information in non-decalcified tissue can be clarified.

  Subsequently, the morphometry of the spine of an 8-month-old mouse was performed using specimens of continuous spinal sagittal slices. Table 1 shows a part of the results of bone morphometry for measuring cancellous bone and L1.

  As a result of the bone morphometry for the cancellous bone, the knockout mouse (homo) had a lower calcification rate than the wild type, and was generally in a state of low turnover bone. This is equivalent to the case of human ectopic ossification.

  Table 2 shows a part of the results of bone morphometry for cortical bone.

  From the results of bone morphometry for cortical bone, knockout mice (homo) showed a thinner spinal cortical bone width than the wild type.

(Diabetes-related)
Further analysis was performed on diabetes.
FIG. 9 is a photomicrograph showing the state of glucagon secretion in the islets of Langerhans in the pancreas of the knockout mouse. A circular image in the photograph is a signal derived from a DAPI-stained nucleus. In addition to the signal derived from this nucleus, glucagon labeled with AlexaFluor 488 could be confirmed, and positive for glucagon in the knockout mouse was confirmed.
In addition, the glucagon level in serum was significantly higher in knockout mice than in wild type mice. Even in patients with posterior longitudinal ligament ossification, serum glucagon levels were higher than in healthy subjects, as in knockout mice.

  FIG. 10 is an HE-stained image of the pancreatic tissue of a knockout mouse. Insulin secretion from the pancreatic tissue was not confirmed, and bleeding from the capillaries, atrophic changes, and a decrease in the number of β cells were confirmed.

  From these results, it was suggested that the knockout mouse is complicated with type I diabetes.

[MiR-340]
It is said that human posterior longitudinal ligament ossification has family-related factors. However, according to the results of analysis of CXCL7 by the patients by the inventors, no abnormalities of CXCL7 exons and SNPs were found. Therefore, the inventors conducted a microRNA array analysis on tissues and blood samples of human posterior longitudinal ligament ossification patients based on the idea that the expression level of CXCL7 protein is affected by microRNA. went.
As a result, it was revealed that miR-340 expression levels were significantly increased in human posterior longitudinal ligament ossification patients compared to healthy subjects.

Real-time PCR
microRNA was extracted using miRCURY RNA Isolation (Exiqon, Vedbaek) according to the protocol attached to the product. RNA integrity was analyzed using an Agilent 2100 Bioanalyser. RNA samples with an integrity value of 9.0 or higher were reverse transcribed. Superscript VILO cDNA Synthesis Kit (Life Technologies, Carlsbad, CA) was used for reverse transcription. Subsequently, real-time PCR was performed by StepOnePlus Real-Time PCR Analysis System (Applied Biosystems, Carlsbad, CA). The probe used for analysis was obtained from Exiqon.
The probe sequences used in real-time PCR are shown below.
Has-miR-340: 5′-uccgucucaguuacuuuauagc-3 ′ (SEQ ID NO: 7)
FIG. 11 shows the expression level of miR-340 in plasma determined by real-time PCR. The expression level of miR-340 in the plasma of human posterior longitudinal ligament ossification patients was significantly higher than that in healthy subjects.

・ In situ hybridization
In situ hybridization was performed using miRCURY LNA microRNA ISH Optimization (Exiqon, Vedbaek, Denmark). Hybridization was performed at 65 ° C.
The sequence of the probe used in in situ hybridization is shown below.
Has-miR-340: 5'-aatcaG (L) t5 (L) aT (L) tG (L) cT (L) ttataa_N (6) _Y-3 '(SEQ ID NO: 8)
In the above sequences, lowercase letters represent DNA, and uppercase letters (L) represent uppercase letters LNA. N (6) represents amino C6 linker and Y represents Alexa Floor 488. 5 represents 5-methylcytidine (5-mC) and 5 (L) represents 5-mC LNA.
FIG. 12 is a photograph showing the results of analyzing the distribution of miR-340 in tissues of the L5-6 portion of knockout mice and the C5-6 portion of human posterior longitudinal ligament ossification patients by in situ hybridization. In FIG. 12, the signals scattered in the form of islands are DAPI-stained images of nuclei. In both the knockout mouse and the posterior longitudinal ligament ossification patient, in addition to the signal derived from the nucleus, a signal derived from the probe labeled with Alexa Floor 488 could be confirmed throughout the tissue.

[CXCL7 protein expression level and posterior longitudinal ligament ossification]
In order to examine the relationship between the expression level of CXCL7 protein and the pathophysiology of posterior longitudinal ligament ossification, patients who are in the continuous (A) pathology among the categorization of posterior longitudinal ligament ossification (see FIG. 6) described above And the expression level of CXCL7 protein in the serum of patients in a mixed (C) pathological condition. Analysis was performed using ELISA Kit (Ray Biotech, Norcross GA). The CXCL7 antibody used was fixed in the well attached to the kit.
The results are shown in FIG. The expression level of CXCL7 in the mixed type (C) patient with a lighter pathological condition was lower than the expression level of CXCL7 in the continuous type (A) patient. This indicates that there is a correlation between the expression level of CXCL7 and the severity of posterior longitudinal ligament ossification.

[Inhibition of CXCL7 in ligament tissue derived from stem cells]
PRELP gene was introduced into equine mesenchymal stem cells to prepare ligament tissue.
CXCL7 protein was suppressed with shRNA-CXCL7 on the obtained ligament tissue. As a negative control, shRNA-GAPDH was used instead of shRNA-CXCL7.
The sequence of shRNA-CXCL7 is shown below.
shRNA-CXCL7: 5'-aaaagaacccattgcaaccaagtttggatccaaacttggttgcaatgggttc-3 '(SEQ ID NO: 9)
The sequence of shRNA-GAPDH is shown below.
shRNA-GAPDH: 5′-aaaacgggaagcttgtcatcaatttggatccaaattgatgacaagcttcccg-3 ′ (SEQ ID NO: 10)
The shRNA-CXCL7 sequence was incorporated into the pLV-H1-CMV-green vector (BiOSETTIA), and shRNA-CXCL7 was introduced into each of the stem cells and the tissue from which the ligament was derived from the stem cells. It introduced similarly about shRNA-GAPDH. The cells after vector introduction were changed to single cells, labeled with BMP-2 antibody, and cells adsorbed with BMP-2 antibody were counted by FACS (n = 3). BMP-2 is an osteoinductive protein and an osteogenic factor. A part of the obtained data is shown in FIG. FIG. 14 (B) shows the measurement results for cells of ligament tissue prepared by introducing the PRELP gene into equine mesenchymal stem cells. FIG. 14 (A) shows the measurement results for the cells of the ligament tissue after knocking down the ligament tissue with shRNA-CXCL7. The results are shown in Table 3.

  BMP-2 positive cells did not change in stem cells and ligament tissue derived from the same stem cells, but when CXCL7 was knocked down with shRNA in the ligament tissue, the value of BMP-2 positive cells increased. Indicated. This means that suppression of CXCL7 in ligament tissue derived from stem cells induces ligament ossification.

[Inhibition of miR-340]
-Calcification staining shRNA-CXCL7 was introduced into mouse osteoblast MC3T3-E1. In addition, miR-340 inhibitor was introduced into mouse osteoblast MC3T3-E1. As a negative control, shRNA-LacZ was used instead of shRNA-CXCL7.
The sequence of shRNA-CXCL7 is identical to the base sequence represented by SEQ ID NO: 9.
shRNA-CXCL7: 5'-aaaagaacccattgcaaccaagtttggatccaaacttggttgcaatgggttc-3 '(SEQ ID NO: 9)
The miR-340 inhibitor used was a commercially available miRCURY LNA Power Inhibitor, has-miR-340 manufactured by EXIQON. has-miR-340 is a nucleic acid that hybridizes with miRNA-340.
The sequence of shRNA-lacZ is shown below.
shRNA-lacZ: 5′-aaaagcagttatctggaagatcaggttggatccaacctgatcttccagataactgc-3 ′ (SEQ ID NO: 11)
The shRNA-CXCL7 sequence was incorporated into a pLV-H1-CMV-green vector (BiOSETTIA), and shRNA-CXCL7 was introduced into MC3T3-E1. miR-340 inhibitor and shRNA-lacZ were similarly introduced.
Next, in order to confirm the phenotype of cell ossification, calcified bone nodules were stained by calcification staining, and the degree of progression of ossification was confirmed.
The results are shown in FIG. No staining was confirmed in control MC3T3-E1 cells (control) into which shRNA had not been introduced. Similarly, staining was not confirmed in MC3T3-E1 cells (sh-LacZ) into which shRNA-LacZ was introduced. On the other hand, in MC3T3-E1 cells (sh-CXCL7) into which shRNA-CXCL7 was introduced, staining was remarkably confirmed and ossification was progressing. MC3T3-E1 cells with miR-340 inhibitor (miR-340 inhibitor) were confirmed to have a smaller staining range than MC3T3-E1 cells (sh-CXCL7) with shRNA-CXCL7. . From these results, it was shown that ossification of cells can be suppressed by suppressing miR-340.

Real-time PCR
Real-time PCR was performed on MC3T3-E1 cells into which shRNA was introduced, and the expression levels of CXCL7, BMP-2, and RANKL (receptor activator of nuclear factor-кB ligand) were determined. RANKL is a factor used as a bone tissue marker.

MC3T3-E1 cells into which shRNA-lacZ was introduced were prepared in the same manner as described in [Suppression of miR-340] above (shRNA-lacZ). Similarly, MC3T3-E1 cells introduced with shRNA-CXCL7 (shRNA-CXCL7), MC3T3-E1 cells introduced with miR-340 (miR-340), MC3T3-E1 cells introduced with miRNA-inhibitor-control (MiRNA-inhibitor-control) and MC3T3-E1 cells (miRNA-inhibitor) into which miRNA-inhibitor was introduced were prepared.
The sequence of miR-340 is the base sequence represented by SEQ ID NO: 6.
miRNA-inhibitor was obtained from Exiqon, (Vedbaek, Denmark).

RNA was extracted from each of the above cells. RNA was extracted using the AllPrep DNA / RNA Mini Kit (Qiagen, Valencia, CA) according to the protocol attached to the product. RNA integrity was analyzed using an Agilent 2100 Bioanalyser. Superscript VILO cDNA Synthesis Kit (Life Technologies, Carlsbad, CA) was used for reverse transcription. Subsequently, real-time PCR was performed by StepOnePlus Real-Time PCR Analysis System (Applied Biosystems, Carlsbad, CA). The probe used for analysis was obtained from Exiqon.
Probes used in the real-time PCR are Taqman probes CXCL7, Mm01347901_g1, BMP-2, Mm01254942_m1, RANKL, Mm00441906_m1, 18S, Mm03928990_g1, RANK, Mm00437132_m1.

  FIG. 16 shows the expression levels of CXCL7, BMP-2, and RANKL determined by real-time PCR. In MC3T3-E1 cells (miRNA-inhibitor) into which miRNA-inhibitor was introduced, a decrease in the expression of CXCL7 was confirmed compared to the control. Both MC3T3-E1 cells (shRNA-CXCL7) introduced with shRNA-CXCL7 and MC3T3-E1 cells (miR-340) introduced with miR-340 showed increased expression levels of BMP-2 and RANKL. It was. From these results, it was shown that suppression of miR-340 suppresses the expression of CXCL7. Moreover, it became clear that suppression of CXCL7 and enhancement of miRNA-340 expression showed the same tendency for the promotion of ossification.

[Analysis of phosphorylation modification in early cells of spinal tissue of knockout mice]
Cells were collected from spinal tissue of knockout mice, proteins were extracted, and peptides were purified. The purified peptide (20-30 pmol) was analyzed using a UltiMate 3000 RSLC nano system (HPLC) pump (Thermo Fisher Scientific, MA, USA) double-focusing sector magnetic mass spectrometer. The acceleration potential was 5 kV. Sample ionization was performed with a 6-KeV Xenon beam, and an analysis sample was prepared by a conventional method. The ion spectrum was analyzed by a linear ion trap mass spectrometer Orbitrap Elite (Thermo Fisher Scientific, MA, USA) and nano-ESI interface. The sample used for micro RPLC-MS / MS analysis was injected into a trap column (nano 75 μm id × 280 μm od; packed with 15 cm of Acclaim PepMap C18). Multiple scans were collected for the LTQ mass range m / z 350-2000 (cycle time 45 seconds) and data was collected.

As a result of phosphorylation modification analysis, phosphorylation was detected for E3 ubiquitin ligase, TRAF3, adiponectin, and TZF-L.
About E3 ubiquitin ligase, it turns out that the 419th serine and the 427th serine of the polypeptide consisting of the 409th to 449th regions in the amino acid sequence represented by SEQ ID NO: 12 are phosphorylated. did.
For TRAF3, the 211th threonine of the polypeptide consisting of the 207th to 214th regions in the amino acid sequence represented by SEQ ID NO: 13 is phosphorylated and the 398th to 422th regions. It was found that the 412th serine and the 413th tyrosine of the polypeptide were phosphorylated.
As for adiponectin, in the amino acid sequence represented by SEQ ID NO: 14, the 64th threonine of the polypeptide consisting of the 62nd to 80th regions is phosphorylated and the 116th to 134th regions. It was found that the 119th serine and 127th threonine of the polypeptide were phosphorylated.
Regarding TZF-L, in the amino acid sequence represented by SEQ ID NO: 15, the 677th serine of the polypeptide consisting of the 676th to 683th region is phosphorylated, and the 1651th to 1672th region. It was found that the 1652th methionine and the 1668th serine of the polypeptide consisting of

FIG. 17 shows an electrophoretic image of TRAF3 and ubiquitin extracted from spinal tissue cells. Lane 1 is for primary cultured primary cells of wild type mice, and lane 2 is for primary cultured primary cells of knockout mice.
The band shift was confirmed as a result shown in FIG. 17, indicating that the E3 ubiquitin ligase of TRAF3 was post-translationally modified. In addition, since the degradation of CXCL7 by E3 ubiquitin ligase was confirmed, the disappearance of CXCL7, the causative protein of posterior longitudinal ligament ossification, is thought to be due to this TRAF3 and E3 ubiquitin ligase.

Next, ubiquitin antibody staining was performed on the spinal tissue of knockout mice and the spinal tissue of human OPLL patients. The results are shown in FIG. A signal of ubiquitin antibody staining was confirmed in an elongated shape as shown in FIG. Signals scattered in islands are DAPI-stained images of nuclei.
From the results shown in FIG. 18, it is considered that ubiquitin is expressed and CXCL7 is degraded in the spinal tissue of the knockout mouse and the spinal tissue of the human OPLL patient.

  The configurations and combinations thereof in the embodiments described above are examples, and the addition, omission, replacement, and other modifications of the configurations can be made without departing from the spirit of the present invention. Further, the present invention is not limited by each embodiment, and is limited only by the scope of the claims.

  Since the spinal ligament ossification model non-human animal of the present invention shows a pathological condition of spinal ligament ossification, screening for a preventive or therapeutic agent for spinal ligament ossification can be performed. It also contributes to the elucidation of the mechanism of spinal ligament ossification.

Claims (16)

  A spinal ligament ossification model non-human animal, wherein the function of CXCL7 gene is deficient or suppressed, or miRNA-340 expression is enhanced.   2. The spinal ligament ossification model non-claim according to claim 1, wherein the CXCL7 gene function deficiency or suppression is due to deletion, insertion, addition, or substitution of a part or all of the CXCL7 gene or an expression control region thereof. Human animals.   The spinal ligament ossification model non-human animal according to claim 1 or 2, wherein the CXCL7 gene is a gene encoding an amino acid sequence represented by SEQ ID NO: 3.   The vertebral ligament ossification model non-human animal according to claim 1, wherein the enhanced expression of miRNA-340 is caused by introduction of miRNA-340.   The spinal ligament ossification model non-human animal according to claim 1 or 2, wherein the miRNA-340 is a polynucleotide comprising an RNA sequence represented by SEQ ID NO: 6.   A diabetes model non-human animal, wherein the function of the CXCL7 gene is deficient or suppressed, or the expression of miRNA340 is enhanced.   A spinal ligament ossification model cell, wherein the function of CXCL7 gene is deficient or suppressed, or miRNA-340 expression is enhanced.   The vertebral ligament ossification model cell according to claim 7, which is a cell of parallel fibrous connective tissue.   A diabetes model cell, wherein the function of CXCL7 gene is deficient or suppressed, or miRNA-340 expression is enhanced.   The spinal ligament ossification model non-human animal according to any one of claims 1 to 5, a cell obtained from the spinal ligament ossification model non-human animal, or the spinal column according to claim 7 or 8. A screening method for a prophylactic or therapeutic agent for spinal ligament ossification characterized by administering a test substance to ligament ossification disease model cells.   The prophylactic or therapeutic agent for spinal ligament ossification according to claim 10, wherein after the test substance is administered, the effect of the test substance as a prophylactic or therapeutic agent is determined using the bone morphology of the lesion as an index. Screening method.   The prophylactic or treatment of spinal ligament ossification according to claim 10 or 11, wherein after the administration of the test substance, the effect of the test substance as a prophylactic or therapeutic agent is determined using the expression of miRNA-340 as an index. Agent screening method.   A method for detecting a ligamentous ossification lesion cell of a spinal ligament characterized by detecting miRNA-340 in a biological sample.   14. The method for detecting vertebral ligament ossification lesion cells according to claim 13, wherein a probe capable of hybridizing with miRNA-340 is used.   A kit for diagnosing spinal ligament ossification, comprising a probe capable of hybridizing with miRNA-340.   A therapeutic agent for spinal ligament ossification, comprising as an active ingredient a nucleic acid capable of hybridizing to miRNA-340, or an expression vector capable of expressing the nucleic acid in the administration subject.