【摘要】 背景:miRNA在细胞分化等众多生理病理过程中起重要调控作用,可调节脂肪来源干细胞成骨分化。目的:筛选成骨分化相关miRNA,分析miRNA在脂肪来源干细胞成骨分化过程中的表达模式。方法:从皮下脂肪组织中分离、培养人脂肪来源干细胞。应用基因芯片技术筛选脂肪来源干细胞成骨诱导前后表达差异显著的miRNA,采用RT-PCR技术检测所筛选的miRNA在成骨诱导第7,14,21天上调/下调的相对表达强度,以ELISA试剂盒检测相应时间点成骨相关蛋白的表达情况。结果与结论:①倒置显微镜下观察,传3代后可获得均一性较高的脂肪来源干细胞,一定诱导条件下具有成骨、成脂、成软骨分化能力。②基因芯片技术筛选出成骨分化前后表达差异变化明显的9个miRNA,其中5个上调,4个下调。③成骨诱导第7天,miR-106a表达上调1.58倍(P<0.05)。第14天时,9个miRNA均表达上调。第21天时,5个miRNA表达上调,4个表达下调。④成骨相关蛋白骨钙素、碱性磷酸酶、Ⅰ型胶原及骨唾液酸蛋白的质量浓度在诱导成骨分化第7天即明显升高,第14天达到峰值,第21天略有下降。更多还原

【Abstract】 BACKGROUND:miRNAs have emerged as important regulators in various physiological and pathological processes of cell differentiation,and can regulate the osteogenic differentiation of adipose-derived stem cells. OBJECTIVE:To screen the osteogenesis-specific miRNAs,and analyze the expression pattern of these miRNAs in osteogenic differentiation of adipose-derived stem cells. METHODS:Adipose-derived stem cells were isolated and cultured from human subcutaneous fat. The osteogenesis-specific miRNAs were screened by gene microarray technique. The relative expression of these miRNAs was analyzed on 7,14,and 21 days by RT-PCR. The osteogenesis-specific proteins were detected on 7,14,and 21 days by enzyme linked immunosorbent assay kit. RESULTS AND CONCLUSION:①The 3rd passage adipose-derived stem cells were homogeneous. Osteogenesis,adipogenesis,and chondrogenesis differentiation of adipose-derived stem cells need specific condition under an inverted microscope. ②Nine osteogenesis-specific miRNAs were picked up by gene microarray technique,five were upregulated and four were downregulated. ③On day 7 in osteogenic differentiation,miR-106a expression was upregulated 1.58 folds(P < 0.05) . On day 14,nine miRNAs were upregulated. On day 21,five miRNAs were upregulated and four were downregulated. ④The concentration of osteogenesis-specific proteins such as osteocalcin,alkaline phosphatase,collagen Ⅰ and bone sialoprotein were increased on day 7,peaked on day 14 and slightly decreased on day 21.更多还原