【摘要】 目的研究大鼠骨髓间充质干细胞(mesenchymal stem cells,MSCs)在体外定向分化为骨骼肌细胞的干预条件。方法取健康4周龄Wistar大鼠MSCs制成细胞悬液接种于培养瓶中,置于37℃、5%CO2恒温培养箱中培养,倒置相差显微镜下观察,细胞布满瓶底即为1代,取第3代MSCs,以5-氮杂胞苷10μmol/L、成肌分化因子0.1ng/ml、转化生长因子β10.1ng/ml、胰岛素样生长因子12ng/ml进行联合诱导,使之分化。诱导后第9天,收集细胞,行MTT比色法、流式细胞仪和免疫组织化学检测鉴定细胞。结果原代培养的MSCs呈贴壁生长,3～5d呈集落样生长,5～7d后有体积不等的多突成纤维细胞、较大的扁平多角形细胞、中等的多角形细胞和较小的三角形细胞。培养12d后,细胞融合,铺满瓶底,MSCs形态变化不明显。联合诱导后,部分细胞死亡,生长缓慢;7d后见细胞明显增殖,体积逐渐增大,呈椭圆形、梭形或不规则形状;14d后,大量增殖的长梭形细胞开始增多;18～22d后,肌管数量增多,体积增大,核数亦明显增多,初生肌管与长梭形成肌细胞呈平行排列,间隔分布。MSCs免疫组织化学法测定CD44反应阳性,胞质呈棕褐颗粒,核周明显,CD34呈阴性反应;诱导后MSCs结蛋白、骨骼肌特异肌球蛋白均为阳性表达。流式细胞仪测定MSCs和诱导后MSCs大部分处于G0/G1期,分别占79.4%、62.9%,而G2/S/M期仅占20.6%、36.1%。根据MTT绘制生长曲线,可见MSCs生长速度明显高于诱导后的MSCs。两种细胞并未达到平台期,都有继续增殖的趋势。结论在体外,MyoD、5-氮杂胞苷可以诱导MSCs向骨骼肌细胞定向分化,并伴有结蛋白和骨骼肌肌球蛋白阳性表达;定向诱导后MSCs增殖期比例大,向骨骼肌细胞分化纯度更高。更多还原

【Abstract】 Objective To explore the in vitro differentiation of the rat mesenchymal stem cells(MSCs) into the skeletal muscle cells induced by the myoblast differentiation factor(MyoD) and 5-azacytidine.Methods The MSCs were taken from the rat bone marrow and the suspension of MSCs was made and cultured in the homeothermia incubator which contained 5% CO2 at 37℃.The cells were observed under the inverted phase contrast microscope daily.The cells spreading all the bottom of the culture bottle were defined as one passage.The differentiation of the 3rd passage of MSCs was induced by the combination of 5-azacytidine,MyoD,transforming growth factor β1,and the insulin-like growth factor 1.Nine days after the induction,the induced MSCs were collected,which were analyzed with the MTT chromatometry,the flow cytometry,and the immunohistochemistry.Results The primarily cultured MSCs grew as a colony on the walls of the culture bottle;after the culture for 5-7 days,the cells were shaped like the fibroblasts,the big flat polygonal cells,the medium-sized polygonal cells,and the small triangle cells;after the culture for 12 days,the cells were found to be fused,spreading all over the bottle bottom,but MSCs were unchanged too much in shape.After the induction by 5-azacytidine,some of the cells died,and the cells grew slowly.However,after the culture for 7 days,the cells grew remarkably,the cell volume increased gradually in a form of ellipse,fusiform or irregularity.After the culture for 14 days,the proliferated fusiform cells began to increase in a great amount.After the culture for 18-22 days,the myotubes increased in number and volume,with the nucleus increased in number,and the newly-formed myotubes and the fusiform myoblst grew parallelly and separately.The immunohistochemistry for MSCs revealed that CD44 was positive in reaction,with the cytoplasm in a form of brown granules.And the nucleus had an obvious border,and CD34 was negative.The induced MSCs were found to be positive for desmin and specific myoglobulin of the skeletal muscle.The flow cytometry showed that most of the MSCs and the induced MSCs were in the stages of G0/G1,accounting for 79.4% and 62.9%,respectively;however,the cells in the stages of G2/S accounted for 20.6% and 36.1%.The growth curve was drawn based on MTT,which showed that MSCs were greater in the growth speed than the induced MSCs.The two kinds of cells did not reach the platform stage,having a tendency to continuously proliferate.Conclusion In vitro,the rat MSCs can be differentiated into the skeletal muscle cells with an induction by MyoD and 5-azacytidine,with a positive reaction for the desmin and the myoglobulin of the skeletal muscle.After the induction,the proliferation stage of MSCs can be increased,with a higher degree of the differentiation into the skeletal muscle.更多还原