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Application of long chain non-coding RNA lnc-HCAR in preparation of bone repair system and bone repair system and preparation method

A long-chain non-coding, bone repair technology, applied in biochemical equipment and methods, DNA/RNA fragments, recombinant DNA technology, etc., can solve bone vascularization and lack of cell viability, repair failure, core part ischemic necrosis, etc. problems, to achieve the effect of promoting vascularization and bone repair, promoting bone repair, and good application prospects

Active Publication Date: 2021-04-20
ARMY MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] In view of this, the purpose of the present invention is to provide a long-chain non-coding RNA lnc- Application of HCAR in the preparation of bone repair system based on endochondral osteogenesis and its bone repair system to solve the problems of vascularization and lack of cell viability in bulk tissue engineering bone

Method used

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  • Application of long chain non-coding RNA lnc-HCAR in preparation of bone repair system and bone repair system and preparation method
  • Application of long chain non-coding RNA lnc-HCAR in preparation of bone repair system and bone repair system and preparation method
  • Application of long chain non-coding RNA lnc-HCAR in preparation of bone repair system and bone repair system and preparation method

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Experimental program
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Effect test

Embodiment 1

[0036] Example 1. Upregulation of lnc-HCAR in the process of endochondral osteogenesis

[0037] 1. Induction of chondrogenic differentiation of bone marrow MSCs followed by induction of hypertrophic chondrogenic differentiation

[0038] The primary culture of bone marrow MSCs was carried out by density centrifugation. When the bone marrow MSCs were fused to about 90%, they were digested with 0.25% trypsin, and then incomplete cartilage induction medium (that is, DMEM low-sugar medium added with 1% ITS+premix , 100mg / mL streptomycin, 100U / mL penicillin, 50μg / mL vitamin C, 40μg / mL proline and 100nM dexamethasone) resuspended and adjusted the cell density to 5.0×10 5 / mL, the resulting cell suspension was divided into 0.5mL cell suspension (2.5×10 5 cells) for aliquoting, and then centrifuged at 500g for 10min to agglomerate the cells. There is no need to discard the supernatant or resuspend the cells after the centrifugation. Induction medium (i.e. 10ng / mL TGF-β3, 1% ITS+premi...

Embodiment 2

[0047] Example 2, lnc-HCAR regulates the expression of Vegfa and Mmp13 in hypertrophic chondrocytes during endochondral osteogenesis

[0048] To investigate whether lnc-HCAR regulates chondrocyte hypertrophy in endochondral osteogenesis, lnc-HCAR was overexpressed and knocked down using lentiviral vectors, respectively. The cell micromass culture method was used to induce hypertrophic differentiation of chondrocytes in vitro, which is an ideal model for the differentiation of chondrocyte hyperplasia into endochondral osteogenesis in vitro. Different groups of MSCs started chondrogenic induction on the second day after centrifugation, and underwent hypertrophy induction on the 14th day after chondrogenic induction. Cell pellets were collected 14 days after induction of hypertrophy. Cell pellets were fixed with paraformaldehyde to extract total RNA, total protein, etc., and stained with Alizarin Red. Then, markers of chondrocyte hypertrophic differentiation were detected using...

Embodiment 3

[0054] Example 3, lnc-HCAR acts as miR-15b-5p molecular sponge, thereby regulating the expression of Vegfa and Mmp13

[0055] 1. lnc-HCAR acts as miR-15b-5p sponge

[0056] To confirm that lnc-HCAR regulates miR-15b-5p in hypertrophic chondrocytes, miR-15b-5p expression was detected in lnc-HCAR overexpression and knockdown hypertrophic chondrocytes.

[0057] image 3 It is because lnc-HCAR can act as miR-15b-5p sponge. image 3 a: Real-time quantitative PCR detection of miR-15b-5p expression changes in hypertrophic chondrocytes after overexpression and silencing of lnc-HCAR. n=3 for each group. image 3 b is a schematic diagram of the vector construction for detecting the binding relationship between miR-15b-5p and lnc-HCAR using dual luciferase reporter genes. image 3 c is the result of dual luciferase reporter gene detection. n=3 for each group. **P<0.01. shNC, control group with silencing lentivirus; shHCAR, lnc-HCAR silencing lentivirus group; NC, overexpression le...

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Abstract

The invention discloses the application of long-chain non-coding RNA lnc-HCAR in preparing a bone repair system based on endochondral osteogenesis, the nucleotide sequence of the lnc-HCAR is shown in SEQ ID NO.1, and the lnc ‑HCAR can promote the expression of Vegfa gene and Mmp13 gene, and can competitively bind miR‑15b‑5p, thereby promoting hypertrophic differentiation of chondrocytes, promoting blood vessel formation in endochondral osteogenesis, and promoting matrix in endochondral osteogenesis remodel. The bone repair system based on long non-coding RNA lnc-HCAR includes lnc-HCAR, gene delivery system, mesenchymal stem cells and porous bone scaffold materials. The invention has good application prospects in tissue engineering bone construction and bone defect repair.

Description

technical field [0001] The invention belongs to the technical field of tissue engineering, and relates to the application of long-chain non-coding RNA lnc-HCAR in preparing a bone repair system, the bone repair system and a preparation method. Background technique [0002] Endochondral ossification is a basic bone formation method and the morphogenesis method of long bones. Endochondral osteogenesis is a process in which cartilage is gradually replaced by bone. During this replacement process, avascular cartilage is gradually transformed into highly vascularized bone tissue. This switch is dependent on the degradation of the cartilage matrix and endochondral angiogenesis. Matrix degradation mediated by Mmp13 expressed by hypertrophic chondrocytes is a prerequisite for invasion of growing cartilage by vascular, precursor osteoclast and osteogenic progenitor cells. Canals and cavities are formed inside the cartilage matrix degraded by Mmp13. Thereafter, blood vessels may i...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61L27/38A61L27/20A61L27/54A61K48/00A61P19/08C12N15/113
CPCA61K48/0066A61L27/20A61L27/3834A61L27/54A61L2300/258A61L2300/64A61L2430/06A61P19/08C12N15/113C12N2310/14
Inventor 董世武白赟张珠
Owner ARMY MEDICAL UNIV
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