Application of long-chain non-coding RNA lnc-HCAR in preparation of bone repair system and bone repair system and preparation method thereof
A long-chain non-coding, bone repair technology, applied in biochemical equipment and methods, DNA/RNA fragments, recombinant DNA technology, etc., can solve repair failure, bone vascularization and lack of cell viability, avascular necrosis at the core, etc. problems, to achieve the effect of promoting bone repair, promoting vascularization and bone repair, and achieving good application prospects.
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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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