Abstract:

Objective To address the clinical challenges of difficulties in self⁃repair of cartilage defects， and to provide new idea for cartilage repair by exploring the roles and potential mechanisms of miR⁃30b in regulating chondrogenic differentiation of rabbit bone marrow⁃derived mesenchymal stem cells（BMSCs）. Methods BMSCs were separated via density gradient centrifugation，and then the cells were identified through flow cytometry and induced bi⁃directional differentiation assay. Changes in cell proliferation ability and HIF⁃1αexpression were detected via CCK⁃8 and Western blotting under different CoCl2 concentrations. Cell line stably expressing miR ⁃ 30b was generated，and the expressions of SOX9 target genes and Wnt/β⁃catenin signaling pathway were detected by Western blotting. Chondrogenic differentiation was induced under hypoxic circumstances. After 21 days，the expressions of collagen Ⅱ and SOX9 were determined by Western blotting and immunohistochemical staining. Results Flowcy⁃ tometry and bi⁃directional differentiation assay showed that the cells highly expressed CD29 and CD34 but lowly expressed CD44 and CD45，and demonstrated good differentiation ability. On the other hand，the CCK⁃8 revealed that hypoxia induced by CoCl2 solution could promote proliferation of BMSCs（P < 0.05）and expression of HIF⁃1α. After 21⁃day chondrogenic induction of the cells stably expressing miR⁃30b in hypoxic circumstances，immunohis⁃ tochemistry and Western blot results demonstrated significant down⁃regulated expressions of collagen Ⅱ and SOX9 as compared with those in the control group，empty vector group and hypoxia group（P < 0.01）. Conclusions miR ⁃ 30b can reduce chondrogenic differentiation of BMSCs in hypoxic circumstances，providing references for epigenetic regulation of stem cells for cartilage defect repair.

Key words:

cartilage defect, miR?30b, bone marrow?derived mesenchymal stem cells, chondrogenic differentiation, hypoxia