摘要
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mi R-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:mi R-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mi R-302阴性对照模拟物mi R-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mi R-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mi R-302过表达实验组碱性磷酸酶活性明显低于对照组(P<0.05)。2mi R-302过表达明显抑制了矿物质沉积钙结节的形成,mi R-302上调实验组橘红色的钙结节明显少于对照组。3mi R-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mi R-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。mi R-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 cells have the capacity of self-renewal and differentiation into certain lineage cells under appropriate conditions. But many mechanisms are unknown until now. OBJECTIVE： To clarify the role of mi R-302 in the regulation of osteogenic and adipogenic differentiation of adipose-derived mesenchymal stem cells. METHODS： Chemically synthesized mi R-302 specific mimics were transfected into adipose-derived mesenchymal stem cells as experimental group. mi R-NC, mi R-302 b negative control mimics, was transfected into another cells 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 mi R-302 upregulation on the adipogenic and osteogenic differentiation of adipose-derived mesenchymal stem cells was analyzed and compared. Western blot assay was used to detect the expression of Runx2 and alkaline phosphatase after regulation of mi R-302. RESULTS AND CONCLUSION：（1） Overexpression of mi R-302 decreased the precipitate and activity of alkaline phosphatase significantly as compared with the control group（P〈0.05）.（2） Overexpression of mi R-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 cells positive for oil red Ostaining 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 mi R-302 can suppress osteogenesis and accelerate adipocytes generation of adipose-derived mesenchymal stem cells. mi R-302 plays a two-way regulatory role to balance the osteogenic and adipogenic differentiation of mesenchymal stem cells.
出处
《中国组织工程研究》
CAS
北大核心
2015年第41期6595-6599,共5页
Chinese Journal of Tissue Engineering Research
基金
江苏省高校自然科学基金(14KJB310021)
江苏省脑病生物信息重点实验室开放课题(JSBl1403)~~
