Application of Semaphorin 4D in preventing and treating bone metabolic diseases

A disease and bone loss technology, applied in bone diseases, gene therapy, medical preparations containing active ingredients, etc., can solve problems such as high fracture risk and decreased bone density, and achieve the effect of reducing drug dosage and preventing osteosclerosis.

Inactive Publication Date: 2014-11-05
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in vivo studies have shown that the Semaphorin 7A gene polymorphism can l

Method used

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  • Application of Semaphorin 4D in preventing and treating bone metabolic diseases
  • Application of Semaphorin 4D in preventing and treating bone metabolic diseases
  • Application of Semaphorin 4D in preventing and treating bone metabolic diseases

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 is aimed at Sema4d mRNA was screened and synthesized according to the Tuschl method Sema4d siRNA.

[0033] Experimental procedure

[0034] The open reading frame of the target gene starts from 75 to 100 bases downstream of the starting codon, and the 19 bases sequence after the "AA" double sequence is searched.

[0035] Analyze the obtained sequence, and select the target gene sequence with a GC ratio between 40-55% as the preferred one. Compare potential sequences with corresponding genomic databases (human, or mouse, rat, etc.), and exclude sequences that are homologous to other coding sequences / ESTs.

[0036] Experimental Results and Conclusion

[0037] we designed Sema4d The siRNA sequence is as follows:

[0038] Sense strand: 5-GUGCCUGGAUAAGAGUAAATT-3 (SEQ ID No: 1); Antisense strand: 5-UUUACUCUUAUCCAGGCACTT-3 (SEQ ID No: 2).

[0039] we designed Sema4d siRNA successfully interfered with Sema4D synthesis at both transcriptional and translation...

Embodiment 2

[0041] Embodiment 2 studies interference silencing Sema4d Effects on osteoclast differentiation

[0042] Experimental procedure

[0043] 1. Isolation and culture of primary bone marrow-derived macrophages

[0044] Take fresh long bones and rinse with PBS containing double antibodies, rinse the bone marrow in the medullary cavity under sterile conditions, pipette into a single cell suspension, separate mononuclear cells with lymphocyte separation medium, rinse with 10ng / ml M-CSF, After resuspended in 10% FBS DMEM, the density of 20,000 cells / well was seeded in a 24-well plate, and the medium was changed every other day. The adherent cells contained abundant bone marrow-derived macrophages.

[0045] 2. Osteoclast Differentiation Induction

[0046] Transfection on the third day of inoculation Sema4d siRNA, and replace the medium with osteoclast induction medium (10ng / ml M-CSF, 100ng / ml RANKL, 10%FBS), change the medium every other day, and observe the formation of osteoclast...

Embodiment 3

[0052] Example 3 Study Interference Silencing Sema4d The effect of osteoclasts on the migration and differentiation of bone marrow mesenchymal cells

[0053] Experimental procedure

[0054] 1. The upper chamber of Transwell is for bone marrow mesenchymal cells, and the lower chamber is for transfection / non-transfection Sema4d siRNA osteoclasts were fixed and stained at 6 hours, and the cells were counted.

[0055] 2. Bone marrow mesenchymal cells with transfection / non-transfection Sema4d Osteoclasts co-cultured with siRNA were added with osteoinductive factors, and the medium was changed every three days.

[0056] 3. Take 7-day co-cultured or conditioned cultured cells for ALP activity measurement.

[0057] 4. Take 14-day co-cultured or conditioned cultured cells for RT-PCR to analyze the expression levels of RUNX2, COL1 and BGLAP.

[0058] 5. Take 21-day co-cultured or conditioned cultured cells for alizarin red staining, and quantitatively analyze their mineralization...

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Abstract

The invention belongs to the field of biomedical medicines and discloses a new target for preventing and treating the bone metabolic disease, namely a brain signal protein family member Semaphorin 4D protein or Sema4d gene, and application of recombinant proteins or antibodies and interfering RNAs all related to the target in preventing or treating bone metabolic diseases.

Description

[0001] technical field [0002] The invention belongs to the field of biomedical drugs, and in particular relates to the application of Semaphorin 4D, a member of the semaphorin family, in the preparation of drugs for treating bone metabolic diseases. Background technique [0003] In healthy bones, the bone remodeling process is precisely regulated by a network of hormones, cytokines, and intercellular interactions. Simply put, osteoclasts that melt and destroy bones on the bone surface and osteoblasts that promote bone formation play a balanced role If this balance is out of balance, you will suffer from bone metabolic diseases, such as increased bone mass (sclerosis), but more often bone loss (osteoporosis) ) and other diseases. [0004] With the acceleration of the aging society, the incidence of osteoporosis is increasing day by day. At present, the incidence rate of the elderly over 60 years old in China is as high as 56%. The basic pathological mechanism of osteoporo...

Claims

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Application Information

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IPC IPC(8): A61K38/17A61K39/395A61K48/00A61K45/00A61P19/08A61P19/10
Inventor 张玉峰边专魏凌飞
Owner WUHAN UNIV
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