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目的:探讨并验证重组miRNA-30a-5p体外转染对人骨髓间充质干细胞(MSCs)向成骨细胞分化的生物学作用。方法:人工重组合成从人骨髓MSCs定向成骨分化差异性表达miRNA基因芯片结果中筛选成骨性表达量显著增高的miRNA-30a-5p。从人体骨髓中分离培养MSCs,成骨诱导分化过程中于体外转染重组miRNA-30a-5p,并通过茜素红S法染色检测钙盐沉积、碱性磷酸酶(ALP)钙钴法染色及ALP比色法定量检测成骨细胞ALP活性,对比观察研究重组miRNA-30a-5p在人骨髓MSCs定向成骨诱导分化过程中的生物学功能。结果:成功分离培养人骨髓MSCs并诱导其成骨定向分化。经体外向MSCs转染miRNA-30a-5p并诱导其成骨定向分化,转染效率为(37.32±2.43)%。分别于诱导培养第14、21天行ALP染色观察、ALP活性测定、茜素红钙盐结节染色检测各组细胞成骨活性,结果显示miRNA-30a-5p mimics转染组MSCs成骨分化特性显著性增强(P<0.05)。结论:miRNA-30a-5p在MSCs定向成骨分化的过程中具有一定的促进增强作用,被转染的MSCs向成骨细胞定向分化表达有所增加,为进一步阐明人骨髓MSCs定向成骨分化的分子生化机制,细胞移植修复治疗骨缺损奠定了理论基础。
Objective: To investigate and verify the biological effects of recombinant miRNA-30a-5p transfection on osteoblastic differentiation of human bone marrow mesenchymal stem cells (MSCs) in vitro. Methods: Recombinant miRNA-30a-5p was screened from miRNA microarray results of osteogenic differentiation and differentiation of human bone marrow MSCs. MSCs were isolated and cultured from human bone marrow. Recombinant miRNA-30a-5p was transfected in vitro during osteogenic differentiation. Calcium salt deposition, Alkaline phosphatase (ALP) calcium and cobalt staining, ALP assay was used to detect the ALP activity of osteoblasts. The biological functions of recombinant miRNA-30a-5p in osteoblast-induced differentiation of human bone marrow MSCs were compared and observed. Results: MSCs were isolated and cultured successfully and their osteogenic differentiation was induced. The miRNA-30a-5p was transfected into MSCs in vitro and its osteogenic differentiation was induced. The transfection efficiency was (37.32 ± 2.43)%. The ALP activity, ALP activity and alizarin red calcium salt nodule staining were used to detect the osteogenic activity of the cells on the 14th and 21st day respectively. The results showed that the osteogenic differentiation of the MSCs in miRNA-30a-5p mimics transfection group Significantly increased (P <0.05). CONCLUSIONS: miRNA-30a-5p can promote the osteogenic differentiation of MSCs. The transfection of MSCs into osteoblasts increases the expression of osteoblasts. To further elucidate the osteogenic differentiation of human bone marrow MSCs Molecular biochemical mechanisms, cell transplantation and repair of bone defects laid the theoretical foundation.