背景：间充质干细胞具有自我更新能力，在一定条件下能够分化为一定谱系的细胞，但很多机制至今未明。目的：探究miR-302b对脂肪间充质干细胞向成脂和成骨分化的调控作用。方法：miR-302模拟物转染作用于脂肪间充质干细胞进行成骨成脂诱导，对照组转染miR-302阴性对照模拟物miR-NC。采用碱性磷酸酶染色及活性分析、茜素红染色、油红O染色和萃取实验观察miR-302上调对脂肪间充质干细胞成骨和成脂分化的影响，以及Western blot检测miR-302上调后成骨分化转录因子Runx2和成骨早期标志物碱性磷酸酶在脂肪间充质干细胞中的表达。结果与结论：①碱性磷酸酶沉淀物在miR-302过表达细胞中产生的量均明显少于对照组，进一步发现miR-302过表达实验组碱性磷酸酶活性明显低于对照组(P <0.05)。②miR-302过表达明显抑制了矿物质沉积钙结节的形成，miR-302上调实验组橘红色的钙结节明显少于对照组。③miR-302过表达实验组油红O染色阳性的细胞数明显高于对照组，进一步表明实验组细胞萃取得到的油红O吸光度值明显上升(P <0.05)。④成骨诱导第6天时成骨分化转录因子Runx2和成骨早期标志物碱性磷酸酶在miR-302过表达的细胞中都有不同程度的下降。⑤以上结果表明 miR-302的上调能够抑制脂肪间充质干细胞的成骨分化，同时促进其向成脂分化。miR-302在间充质干细胞向成脂和成骨分化平衡发挥了双向调控作用。%BACKGROUND:Mesenchymal stem cels have the capacity of self-renewal and differentiation into certain lineage cels under appropriate conditions. But many mechanisms are unknown until now. OBJECTIVE:To clarify the role of miR-302 in the regulation of osteogenic and adipogenic differentiation of adipose-derived mesenchymal stem cels. METHODS:Chemicaly synthesized miR-302 specific mimics were transfected into adipose-derived mesenchymal stem cels as experimental group. miR-NC, miR-302b negative control mimics, was transfected into another cels as control group. By the experiments of alkaline phosphatase staining, alkaline phosphatase activity assay, alizarin red staining, oil red O staining and extraction test, the effect of miR-302 upregulation on the adipogenic and osteogenic differentiation of adipose-derived mesenchymal stem cels was analyzed and compared. Western blot assay was used to detect the expression of Runx2 and alkaline phosphatase after regulation of miR-302. RESULTS AND CONCLUSION: (1) Overexpression of miR-302 decreased the precipitate and activity of alkaline phosphatase significantly as compared with the control group (P < 0.05). (2) Overexpression of miR-302 inhibited the formation of mineral deposits and calcium nodules, and the number of calcium nodules in the experimental group was significantly lower than that in the control group (P < 0.05). (3) The number of cels positive for oil red O staining was significantly higher in the experimental group than the control group, which further showed the absorbance values of oil red O staining in the experimental group obtained in the extraction test were significantly increased (P < 0.05). (4) At 6 days of osteogenic induction, the expressions of Runx2 and alkaline phosphatase in the experimental group were decreased to different extents. These findings indicate that overexpression of miR-302 can suppress osteogenesis and accelerate adipocytes generation of adipose-derived mesenchymal stem cels. miR-302 plays a two-way regulatory role to balance the osteogenic and adipogenic differentiation of mesenchymal stem cels.
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