不同代数的成人骨髓间充质干细胞体外转化为神经细胞的实验研究

目的　探讨不同代数的成人骨髓间充质干细胞 ( h MSCs)体外向神经元细胞转化的效率 ,为骨髓间充质干细胞应用于临床提供可靠的实验数据。方法　采用 β-巯基乙醇做为诱导剂 ,选用第 2、4、6、8代 h MSCs在体外诱导 6 h后 ,用细胞化学及免疫组织化学检测神经元细胞、星形胶质细胞标记蛋白的表达。结果　第 2、4、6代h MSCs诱导后胞浆中均可见深蓝色的颗粒状尼氏体 ,第 8代 h MSCs诱导 6 h后胞浆中未见明显的深蓝色尼氏体结构。不同代数的 h MSCs经诱导 6 h后均表达 NSE、NF- M,不表达 GFAP;第 2、4、6代的阳性率无显著性差异 ( P>0 .0 5 ) ,第 8代与第 2、4、6代的阳性率有显著性差异 ( P<0 .0 5 )。结论　 β-巯基乙醇在体外可定向诱导 h MSCs转化为神经元细胞 ,第 2、4、6代的阳性率明显高于第 8代。

Experimental study on neuro-differentiation of human bone marrow mesenchymal stem cells in vitro

Objective To study efficiency and regularity of inducing human bone marrow mesenchymal stem cells (hMSCs) to neuron in vitro by 2-mercaptoethanol(2-ME).Methods hMSCs were induced by 2-ME in serum-free medium for 6 hours.Induction media were removed and transferred to neuronal induction maintenance media for 6 days.The expression of neural or glia specific markers was evaluated by cytochemistry staining,immunohistochemistry staining and Western blot.Results After induction for 6 hours,hMSCsdisplayed neuronal morphologies such as pyramidal cell bodies and processes formed extensive networks.The induced hMSCs expressed neuro-specific marker such as neuro-specific enolase (NSE) and Neurofilament (NF),but could not express glia fiber acid protein (GFAP),which is astrocytes specific marker.We could discover Nissl bodies in differentiated cells.Nestin was detectable at 6h,and expression levels of hMSCs-derived neurons decreased with time by western blot.By 6 daysafter induction there had been no detectable nestin expression in any hMSCs-derivedneurons.But MAP-2,a marker of mature neuron was not detectable at that time, but was detectable at 3d,and persisted through 6 days.Conclusion hMSCs can differentiated first into nerve stem cells,and then differentiated into neurocyte.