JP2019024462A - Synovitis marker and use thereof - Google Patents

Abstract

To provide a technique to conveniently determine whether a subject contracts synovitis.SOLUTION: A synovitis marker comprises V-Set And Transmembrane Domain Containing 4(VSTM4) gene or protein coded by VSTM4 gene.SELECTED DRAWING: None

Description

  The present invention relates to synovitis markers and uses thereof. More specifically, the present invention relates to a synovitis marker, a synovitis diagnostic kit, a data collection method for diagnosing whether or not a patient suffers from synovitis, and a synovitis therapeutic agent.

  Osteoarthritis, rheumatoid arthritis, and the like are diseases in which pain and swelling occur in the joint. Each disease has similar symptoms in the joints, but there are differences in the cause and how the disease progresses, and the treatments are also different.

  Specifically, osteoarthritis is a disease in which the cartilage of the joint is damaged mainly due to the burden on the joint and excessive use of the joint, and the symptoms progress from there. For this reason, measures such as keeping the function of the joint by forging muscles around the joint by exercise therapy while softening the symptoms by drug therapy are taken. In some cases, braces are used to protect the joints. On the other hand, rheumatoid arthritis is a disease caused by a strong relationship with immune abnormalities. In the treatment of rheumatoid arthritis, measures such as administering a drug that suppresses immune abnormalities are taken.

  With an aging society, the number of osteoarthritis patients continues to increase. However, there is no marker for grasping the disease state. It has been suggested that synovitis may be involved in the progression of osteoarthritis and pain.

  By the way, V-Set And Transmembrane Domain Containing 4 (VSTM4) is a gene located on the long arm of human chromosome 10, but its function is not well understood.

  In recent years, it has been reported that a peptide (Peptide Lv) generated from a protein encoded by the VSTM4 gene is a neuropeptide that controls expression of an L-type voltage-dependent calcium channel present in a photoreceptor (for example, , See Non-Patent Document 1.)

Shi L., et al., Identification of Peptide lv, a novel putative neuropeptide that regulates the expression of L-type voltage-gated calcium channels in regulators., PLoS One, 7 (8), e43091, 2012.

  Conventionally, there has been no technique for simply determining whether or not a patient suffers from synovitis. Then, an object of this invention is to provide the technique which determines easily whether it is suffering from synovitis.

The present invention includes the following aspects.
[1] A synovitis marker comprising a VSTM4 gene or a protein encoded by the VSTM4 gene.
[2] A synovitis diagnostic kit comprising a primer for amplifying the cDNA of the VSTM4 gene, a probe that specifically hybridizes to the mRNA of the VSTM4 gene, or a specific binding substance for the protein encoded by the VSTM4 gene .
[3] Data collection for diagnosing whether or not the patient is suffering from synovitis, comprising the step of measuring the expression level of a VSTM4 gene or a protein encoded by the VSTM4 gene in a patient-derived biological sample Method.
[4] A therapeutic agent for synovitis comprising a substance that controls signal transduction by the VSTM4 protein.

  ADVANTAGE OF THE INVENTION According to this invention, the technique which determines easily whether it is suffering from synovitis can be provided.

(A)-(c) is a graph which shows the result of Experimental example 1. FIG. 10 is a graph showing the results of Experimental Example 2. 10 is a graph showing the results of Experimental Example 3.

[Synovitis marker]
In one embodiment, the present invention provides a synovitis marker consisting of a VSTM4 gene or a protein encoded by the VSTM4 gene. By using the marker of this embodiment, it becomes possible to easily diagnose synovitis. Hereinafter, in the present specification, a protein encoded by the VSTM4 gene may be referred to as a VSTM4 protein.

  As will be described later in the Examples, the inventors found that the expression level of the VSTM4 gene in the synovial tissue significantly increases when synovitis develops, as a result of studies using multiple types of synovitis model mice. Revealed. Also, in the study using synovial tissue of knee osteoarthritis patients, it was revealed that the onset of arthritis and the increase in the expression level of the VSTM4 gene are significantly correlated.

  This embodiment can also be said to provide use of the VSTM4 gene or VSTM4 protein as a synovitis marker.

[Synovitis diagnostic kit]
In one embodiment, the present invention comprises a primer for amplifying VSTM4 gene cDNA, a probe that specifically hybridizes to VSTM4 gene mRNA, or a specific binding substance for VSTM4 protein. Provide kit. The kit of this embodiment can easily diagnose synovitis in patients or livestock.

  It is preferable that the kit of this embodiment detects a gene or protein of a species to be diagnosed as a VSTM4 gene or a VSTM4 protein. For example, when the diagnosis target is human, it is preferable to detect human VSTM4 gene or human VSTM4 protein. The livestock is not particularly limited, and examples thereof include cows, horses, mice and the like.

(Primer set)
The primer set is not particularly limited as long as it can amplify the cDNA of the VSTM4 gene of the species to be diagnosed.

(probe)
The probe is not particularly limited as long as it specifically hybridizes to the mRNA of the VSTM4 gene of the species to be diagnosed. The probe may be fixed on a carrier to constitute a DNA microarray or the like.

(Specific binding substance)
Examples of the specific binding substance include antibodies, antibody fragments, aptamers and the like. The antibody may be prepared, for example, by immunizing an animal such as a mouse with the VSTM4 protein of the species to be diagnosed or a partial peptide thereof as an antigen. Alternatively, it can also be prepared by screening an antibody library such as a phage library. Examples of antibody fragments include Fv, Fab, scFv and the like. The above antibody or antibody fragment may be polyclonal or monoclonal.

  An aptamer is a substance having a specific binding ability to a labeling substance. Examples of aptamers include nucleic acid aptamers and peptide aptamers. Nucleic acid aptamers having specific binding ability to the VSTM4 protein can be selected by, for example, the systematic evolution of ligand by exponential enrichment (SELEX) method. Peptide aptamers having specific binding ability to the VSTM4 protein can be selected by, for example, the two-hybrid method using yeast.

  The specific binding substance is not particularly limited as long as it can specifically bind to the VSTM4 protein, and may be a commercially available one. The specific binding substance may be fixed on a carrier to constitute a protein chip or the like.

[Data collection method]
In one embodiment, the present invention provides data for diagnosing whether or not the patient suffers from synovitis, comprising measuring the expression level of VSTM4 gene or VSTM4 protein in a patient-derived biological sample. Provide collection methods. Note that the data collection method does not include a step of judgment by a doctor.

  In the method of the present embodiment, examples of the biological sample include synovial tissue, joint fluid, serum, plasma, and the like. Moreover, the measurement of the expression level of VSTM4 gene in a biological sample can be performed by, for example, quantitative RT-PCR, DNA microarray analysis, and the like. Moreover, the measurement of the expression level of VSTM4 protein in a biological sample can be performed by, for example, immunohistochemical staining, ELISA, Western blotting, or the like.

  A higher expression level of the VSTM4 gene or VSTM4 protein than the expression level of the VSTM4 gene or VSTM4 protein in a healthy subject indicates that the patient suffers from synovitis.

[Synthetic agent for synovitis]
In one embodiment, the present invention provides a therapeutic agent for synovitis comprising a substance that controls signal transduction by VSTM4 protein. The substance that controls signal transmission by the VSTM4 protein may be, for example, a substance that increases or decreases the expression level of the VSTM4 protein, or a substance that activates or inhibits the function of the VSTM4 protein.

  More specifically, a compound that increases or decreases the expression level of the VSTM4 gene or protein, an inhibitory RNA for the VSTM4 gene, a VSTM4 protein or a fragment thereof, a specific binding substance that inhibits the function of the VSTM4 protein, and the like. Here, examples of the inhibitory RNA include siRNA and shRNA. Specific binding substances that inhibit the function of the VSTM4 protein include neutralizing antibodies against the VSTM4 protein.

  Next, although an experiment example is shown and this invention is demonstrated further in detail, this invention is not limited to the following experiment examples.

[Experimental Example 1]
(Study using synovitis mouse model 1)
Using a synovitis mouse model, the relationship between the onset of synovitis and the expression level of the VSTM4 gene was examined. As a synovitis mouse model, a synovitis model in which the patella of a normal mouse (C57BL / 6J) was permanently dislocated was used.

  RNA was extracted from the synovial tissue of synovitis model mice (n = 10) 7 days after dislocation of the patella, and the expression level of VSTM4 gene mRNA was measured by quantitative RT-PCR. For comparison, the expression level of TNF-α gene mRNA, which is an inflammatory cytokine, was also measured by quantitative RT-PCR. As a control, RNA was extracted from the synovial tissue of normal mice and the same operation was performed (n = 10). In addition, the expression levels of the VSTM4 gene and the TNF-α gene were normalized using the expression levels of the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene, which is a housekeeping gene.

  For PCR of the mouse VSTM4 gene, the sense primer shown in SEQ ID NO: 1 and the antisense primer shown in SEQ ID NO: 2 were used. Moreover, the sense primer shown to sequence number 3 and the antisense primer shown to sequence number 4 were used for PCR of a mouse | mouth TNF- (alpha) gene. Moreover, the sense primer shown to sequence number 5 and the antisense primer shown to sequence number 6 were used for PCR of a mouse | mouth GAPDH gene.

  1A to 1C are graphs showing the examination results. FIG. 1 (a) is a graph showing the expression level of the VSTM4 gene. FIG. 1 (b) is a graph showing the expression level of the TNF-α gene. 1 (a) and 1 (b), the difference between the two groups was tested using the Mann-Whitney U test, and P <0.05 was considered significant. In FIG. 1 (a) and (b), “*” indicates that there is a significant difference.

  FIG. 1C is a graph showing the correlation between the expression level of the VSTM4 gene and the expression level of the TNF-α gene. Correlation was examined by calculating Pearson's correlation coefficient, and P <0.05 was considered significant.

  As a result, it was revealed that the synovitis model showed a significant increase in the expression of the VSTM4 gene and the TNF-α gene compared to the control. Moreover, it became clear that a significant correlation was observed between the expression level of the VSTM4 gene and the expression level of the TNF-α gene.

  The above results indicate that it is possible to determine whether or not the patient suffers from synovitis by measuring the expression level of the VSTM4 gene or protein.

[Experiment 2]
(Study using synovitis mouse model 2)
Subsequently, the relationship between the onset of synovitis and the expression level of the VSTM4 gene was examined using a synovitis mouse model different from Experimental Example 1. As a synovitis mouse model, a knee osteoarthritis spontaneous mouse (STR / Ort, Nippon Charles River) was used. STR / Ort mice develop mild synovitis at about 3 months of age, moderate synovitis at 6 months of age, and severe synovitis at 18 months of age.

  RNA was extracted from the synovial tissue of 6-month-old and 18-month-old synovitis model mice (n = 10), and the expression level of VSTM4 gene mRNA was measured by quantitative RT-PCR. As a control, RNA was extracted from the synovial tissue of normal mice (C57BL / 6J), and the same operation was performed (n = 10). In addition, the expression level of the VSTM4 gene was normalized using the expression level of the GAPDH gene. As primers for amplifying each gene, the same primers as in Experimental Example 1 were used.

  FIG. 2 is a graph showing the results. The difference between the two groups was tested using Mann-Whitney U test, and P <0.05 was considered significant. In FIG. 2, “*” indicates that there is a significant difference. As a result, it was clarified that the VSTM4 gene expression was significantly increased in the synovitis model as compared with the control.

  The above results further support that it is possible to determine whether or not the patient suffers from synovitis by measuring the expression level of the VSTM4 gene or protein.

[Experiment 3]
(Study using human patient samples)
This experimental example was submitted to the Kitasato University School of Medicine / Hospital Ethics Committee under the title “Exploratory research on synovial / osteochondral tissue-derived pain in orthopedic diseases” and was approved (Approval Number: B3). -113).

First, synovial tissue was collected from 137 knee osteoarthritis patients during total knee arthroplasty. The sexes of the patients were 29 males and 108 females. The patient's age was 72.8 ± 8.2 years. The patient's Body Mass Index was 26.1 ± 0.4 kg / m ² .

  Subsequently, RNA was extracted from each collected synovial tissue, and the expression level of VSTM4 gene mRNA was measured by quantitative RT-PCR. Moreover, the expression level of mRNA of TNF-α gene, which is an inflammatory cytokine, was also measured. In addition, the expression levels of the VSTM4 gene and the TNF-α gene were normalized using the expression level of the GAPDH gene. Subsequently, the correlation between the expression level of VSTM4 gene mRNA and the expression level of TNF-α gene mRNA was examined.

  For PCR of the human VSTM4 gene, the sense primer shown in SEQ ID NO: 7 and the antisense primer shown in SEQ ID NO: 8 were used. For the PCR of the human TNF-α gene, the sense primer shown in SEQ ID NO: 9 and the antisense primer shown in SEQ ID NO: 10 were used. For PCR of the human GAPDH gene, the sense primer shown in SEQ ID NO: 11 and the antisense primer shown in SEQ ID NO: 12 were used.

  FIG. 3 is a graph showing the correlation between the expression level of the VSTM4 gene and the expression level of the TNF-α gene. Correlation was examined by calculating Pearson's correlation coefficient, and P <0.05 was considered significant.

  As a result, it has been clarified that the expression level of the VSTM4 gene in the synovial tissue of knee osteoarthritis patients is significantly correlated with the expression level of the TNF-α gene.

  This result further supports that it is possible to determine whether or not a human suffers from synovitis by measuring the expression level of the VSTM4 gene or protein in humans.

  ADVANTAGE OF THE INVENTION According to this invention, the technique which determines easily whether it is suffering from synovitis can be provided.

Claims (4)

  A synovitis marker comprising a protein encoded by V-Set And Transmembrane Domain Containing 4 (VSTM4) gene or VSTM4 gene. Primers for amplifying the VSTM4 gene cDNA;
A probe that specifically hybridizes to the mRNA of the VSTM4 gene, or a specific binding substance to the protein encoded by the VSTM4 gene,
A synovitis diagnostic kit comprising:   A data collection method for diagnosing whether or not the patient is suffering from synovitis, comprising the step of measuring the expression level of a VSTM4 gene or a protein encoded by the VSTM4 gene in a patient-derived biological sample.   A therapeutic agent for synovitis comprising a substance that controls signal transduction by VSTM4 protein.

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