人骨髓间充质干细胞向胆碱能神经元定向分化的实验研究

Differentiation of Human Bone Marrow Mesenchymal Stem Cells into Cholinergic Neurons in vitro

目的探讨人骨髓间充质干细胞(BMMSCs)体外定向分化为神经元的潜能。方法体外分离和扩增人BMMSCs。利用扩增后的第2代BMMSCs分为诱导组和对照组。诱导组的细胞经预处理和预诱导后在含全反式维甲酸(ATRA)和音速波状蛋白(Shh)的完全培养基中诱导5 h,对照组不加任何诱导剂。诱导后观察细胞形态变化,采用免疫细胞化学染色法检测神经元样细胞特异标志。结果诱导组BMMSCs经诱导后均能分化为具有典型神经元形态的细胞,并表达神经元特异性烯醇化酶[NSE,(61.00±3.63)%]、胶质纤维酸性蛋白[GFAP,(14.42±2.74)%],且有相当部分神经元样细胞表达胆碱乙酰转移酶[ChAT,(9.92±1.29)%];对照组BMMSCs细胞形态无明显变化,上述特异性标志物表达也均为阴性。结论BMMSCs可跨胚层分化为神经元样细胞和胆碱能神经元样细胞。

Objective To explore the differentiation potentiality of human bone marrow mesenchymal stem cells （BMMSCs） into eholinergie neurons in vitro. Methods The human BMMSCs were isolated from fresh bone marrow by a method of density gradient eentrifugation, then amplified and cultivated Lu a total medium of DMEM/F12 containing 10% FBS. BMMSCs were divided into two groups in the second passage. In the induction group, BMMSCs were pretreated with a total mediem consisting of 40 mg/L WHI-P131 for 48 h and preindueed with 10 ng/mL basic fibroblast growth factor （bFGF） for 24 h, then induced with 1 p_mol/L alhransretinoie acid （ATRA） and 10 ng/mL sonic hedgehog （Shh） for 5 h. On the other hand, no inductive agents were added in the control group. After the induction, BMMSCs were observed using phase-contrast microscopy and detected by neuron specific enolase （NSE）, glia fibrillary acid protein （GFAP） and choline aeetyhransferase （CHAT） immunoeytoehemistry, then the percentages of NSE, GFAP and ChAT positive cells were calculated respectively. Results Under induction conditions, most of BMMSCs resumed typical neuronal morphology, and the percentages of NSE, GFAP and ChAT positive cells were （61.00±3.63）%, （14.42±2.74）% and （9.92±1.29）%respectively. But in the control group, BMMSCs remained unehange morphologically and none of them expressed NSE, GFAP and CHAT. Conclusions Human BMMSCs can be induced to differentiate into neuron- like and eholinergie neuron-like cells in vitro.