【摘要】 为了探讨miR-181a对牛骨骼肌卫星细胞分化的影响,试验将分离培养的牛骨骼肌卫星细胞接种在6孔板中,待细胞汇合度为70%时,用PEI转染miR-181a的模拟物miR-181a-M、抑制剂miR-181a-I、模拟物对照miR-181a-M-NC和抑制剂对照miR-181a-I-NC进入牛骨骼肌卫星细胞中,在0,1,2,3天用Western-blot检测肌肉有关分化蛋白MyoD、MyoG和MYH3以及靶基因HOX-A11蛋白的表达量,用免疫荧光法检测细胞肌管的数量。结果表明:在牛骨骼肌卫星细胞中,转染miR-181a模拟物的牛骨骼肌卫星细胞,有关肌肉分化蛋白质如MyoD、MyoG和MYH3的表达量明显增加,细胞肌管数量显著增多,进一步证实miR-181a促进细胞的分化。在双荧光素酶报告系统中,miRNA-181a和HOX-A11基因的3’-UTR结合时荧光素酶活性明显下降,miRNA-181a和突变的3’-UTR结合时荧光素酶活性没有下降。说明HOX-A11是miR-181a的靶基因,miR-181a对牛骨骼肌卫星细胞的分化具有促进作用。更多还原

【Abstract】 The aim of the present study was to investigate the effect of miR-181a on the differentiation of bovine skeletal muscle satellite cells.The bovine skeletal muscle satellite cells were isolated and seeded in six-well plates. When the cell confluence reached 70%,the miR-181a-M,miR-181a-I,miR-181a-M-NC and miR-181a-I-NC were transfect into the bovine skeletal muscle satellite cells by using the PEI. The expression of relative muscle differentiated proteins MyoD,MyoG,MYH3 and HOX-A11 and the myotube number were detected by Western-blot and mmunofluorescence at the 0,1 st,2 nd and 3 rd day after tranfection. The results showed that the bovine skeletal muscle satellite cells transfected with miR-181a mimics were significantly increased in the expression of muscle differentiation proteins such as MyoD,MyoG and MYH3,and the number of myocardium transfected with miR-181a mimics was significantly higher than that of others. It was confirmed that the cell differentiation could be promoted by the miR-181a. In the dual luciferase reporter system,when the 3’-UTR of the HOX-A11 gene was bound to miRNA-181a,the luciferase activity was significantly decreased. But the luciferase activity did not decrease when the mutated 3’-UTR was bound to miRNA-181a. The results further indicated that the HOX-A11 was the target gene of miR-181a. In general,the miR-181a could play a role in promoting the differentiation of bovine skeletal muscle satellite cells.更多还原