大鼠骨髓间充质干细胞培养、鉴定及神经样细胞分化

Neuron-like cells differentiated from rat bone marrow mesenchymal stem cells which were cultured and identified in vivo

目的建立稳定的大鼠骨髓间充质干细胞(BMSCs)体外培养、纯化、扩增的实验体系,并进行细胞表面抗原的鉴定及定向诱导分化检测。方法采用全骨髓贴壁培养法分离、纯化大鼠BMSCs;观察细胞形态,采用细胞免疫化学染色及流式细胞术检测细胞表面CD90、CD29、CD34和CD45表达;分别使用β-巯基乙醇及碱性成纤维细胞生长因子诱导细胞向神经样细胞分化,采用Western印迹法检测诱导后神经标志蛋白[神经巢蛋白(Nestin)、神经元特异烯醇化酶(NSE)、胶质纤维酸性蛋白(GFAP)]表达。结果培养至第3代的BMSCs,CD90、CD29表达阳性,CD34和CD45表达阴性;经诱导后,神经标志蛋白表达显著增高(P<0.05)。结论全骨髓贴壁培养法可分离、培养得到高纯度、具备相关生物学特性的BMSCs,且经诱导可定向分化为神经样细胞。

Objective To establish a stable experimental system to culture, purify, amplify and identify rat bone marrow mesenchy- mal stem cells （BMSCs） in vitro. Methods BMSCs were isolated from rats by wall sticking method. The cells＇ morphology was observed, the expressions of CD90, CD29, CD34 and CD45 were detected by immunocytochemical staining and flow cytometry. Beta mercapto ethanol and basic fibroblast growth factor were separately used to induce differentiation of BMSCs into neuron-like cells. The expressions of neural marker proteins such as Nestin, NSE and GFAP were detected by Western blot. Results The expressions of CD90 and CD29 were positive in cul- tured BMSCs for the third generation, and the expressions of CD34 and CD45 were negative. Compared with the non-induced BMSCs, the expressions of Nestin, NSE and GFAP of induced BMSCs were obviously higher （ P〈0.05 ）. Conclusions A stable experimental method is successfully established to isolate and culture BMSCs which could also be induced into neuron-like cells. The experimental system will be the basis for further research.