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目的 体外诱导大鼠骨髓间质干细胞 (rMSC)分化为神经细胞。方法 用SD大鼠股骨骨髓细胞体外培养。用绞股蓝总甙加入无血清L DMEM诱导MSC分化为神经细胞。免疫细胞化学鉴定有神经元烯醇化酶 (NSE)、神经干细胞标志物巢蛋白 (nestin)和胶质纤维酸性蛋白 (GFAP)的表达。结果 大鼠骨髓间质干细胞在体外扩增 5～ 2 2代 ,对照组不加任何诱导剂 ,MSC可分化为神经样细胞 (5 3 1%± 4 3% )。加入绞股蓝诱导剂诱导 1～ 5h ,MSC形态转变为典型的神经样细胞(90 0 %± 4 6 % )。免疫细胞化学染色显示诱导出的神经样细胞 ,NSE、nestin、GFAP表达阳性。继续培养 12～ 19d ,5d后对照组所有细胞逐渐死亡。绞股蓝组持续诱导 12～ 19d后神经样细胞形态完好 ,大部分细胞变为圆形并聚集成团 ,NSE、nestin、GFAP表达阳性。如果换回正常培养液 ,神经样细胞可逆转回MSC并传代。结论 骨髓本身可能存在神经干细胞。大鼠骨髓间质干细胞用中成药绞股蓝诱导可分化为多种形态的神经样细胞。
Objective To induce rat bone marrow mesenchymal stem cells (rMSCs) to differentiate into neural cells in vitro. Methods SD rat femur bone marrow cells were cultured in vitro. The serum-free L-DMEM was used to induce differentiation of MSCs into neural cells with Gynostemma pentaphyllum. Immunocytochemistry identified the expression of neuronal enolase (NSE), nestin, a neural stem cell marker, and glial fibrillary acidic protein (GFAP). Results Rat bone marrow mesenchymal stem cells were expanded in vitro for 5 to 22 passages. In the control group, no inducer was added. MSCs could differentiate into neuron-like cells (5 3 1% ± 4 3%). After induction with Gynostemma inducer for 1–5 h, the morphology of the MSCs was converted to typical neuron-like cells (90% ± 46%). Immunocytochemical staining showed that induced neuron-like cells were positive for NSE, nestin, and GFAP. After 12 to 19 days, all cells in the control group died gradually after 5 days. In the Gynostemma group, the appearance of nerve-like cells was intact after 12 to 19 days of continuous induction. Most of the cells became round and clustered, and NSE, nestin, and GFAP were positive. If the normal culture fluid is changed back, the nerve-like cells can be reversed back to the MSC and passaged. Conclusion Neural stem cells may be present in the bone marrow itself. Rat bone marrow mesenchymal stem cells can be differentiated into various forms of neuron-like cells induced by the Chinese patent medicine Gynostemma.