脐带间充质干细胞的分离培养及诱导分化为多巴胺能样神经元

Isolation of human umbilical cord mesenchymal stem cells and their differentiation into dopaminergic neurons

目的 探索脐带间充质干细胞（MSCs）的分离培养及将其诱导分化为多巴胺能神经元的方法。方法 分离脐带间充质组织，Ⅳ型胶原酶消化分离脐带MSCs．原代培养于含10％FBS的DMEM／F12培养基中，观察细胞形态并应用流式细胞仪检测细胞表面标志物和细胞周期，CCK8法绘制细胞生长曲线；采用二步法诱导分化P3代脐带MSCs，培养3、6、9d后终止诱导，应用免疫荧光染色和Westernblotting检测分化后细胞酪氨酸羟化酶（TH）、神经元特异性烯醇化酶ⅢSE）的表达。结果 分离培养的脐带MSCs形态呈相对均一的成纤维样细胞，平行排列或旋涡状生长。P3代细胞CD29、CD44、CD73、CD90、CDl05、CDl66表达阳性，而CD34、CD45、CDl9、CD31、HLA—DR表达阴性。对数生长期细胞倍增时间为48h，处于GO～G1期细胞占91．13％；诱导分化后细胞多数为两级．形态与神经元相似．免疫荧光染色检测结果显示诱导9d时细胞NSE、TH染色阳性率分别为19．5％和8．9％，Weston blotting检测结果显示诱导6d时细胞NSE表达明显，TH仅有弱表达，诱导9d时TH、NSE均表达明显。结论 脐带间充质组织中能分离出MSCs，且能分化成多巴胺能神经元。

Objective To investigate the methods of isolation of human umbilical cord mesenchymal stem cells （MSCs） in Wharton＇sjelly and the differentiation ofMSCs into dopaminergic neurons. Methods The umbilical cord mesenchymal tissue was scraped off from the Wharton＇ s jelly, and then, collagenase IV was employed to isolate the MSCs. The isolated ceils were primarily cultured in DMEM/F12 medium containing 10% FBS. Inverted microscopy was used to observe the cytomorphology, and flow cytometry was employed to detect the cell surface antigens and the cell cycle. We evaluated the cell viability using CCK8 kit. Two-step method was employed to induce the MSCs of the P3 generation to differentiate into dopaminergic neurons, and immunocytochemistry and Western blotting were used to detect the expressions of neuron specific enolase （NSE） and tyrosine hydroxylase （TH） 3, 6 and 9 d after the induction. Results The isolated MSCs showed fibroblast-like shape, with parallel arrangement and vortex-like growth. MSCs of the P3 generation expressed CD73, CD29, CD44 and CD105, but did not express CD34, CD45, CD106 and HLA-DR. The doubling time in the exponential phase was at the 48^th h of culture, and 91,13% cells were under G0-G1. These cells had similar morphology of the neurons. The immunocytochemical assay showed that the NSE and TH positive rates were 19.5% and 8.9% on the 9^th d of induction; and Western blotting showed that MSCs obviously expressed NSE and weakly expressed TH. Conclusion MSCs can be isolated from the umbilical cord mesenchymal tissue, and be induced to differentiate into dopaminergic neurons in vitro.