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目的:建立诱导成人骨髓间质干细胞(bone marrow mesenchymal stem cells,BMSCs)向神经干细胞分化的体外培养方法,为细胞移植修复神经系统损伤提供种子细胞。方法:原代贴壁培养成人BMSCs并传代培养至第3代时,将细胞接种于铺有琼脂糖的培养瓶,用神经干细胞培养基(含B27,EGF,bFGF)抗贴壁诱导培养。将诱导培养的神经球(neurospheres)传代扩增。用免疫荧光染色和蛋白质印迹法,对贴壁培养的BMSCs和抗贴壁诱导培养的神经球细胞表达神经干细胞标志蛋白的水平进行检测和比较。结果:抗贴壁诱导培养可使BMSCs形成神经球,初级神经球传代可扩增形成次级神经球。神经球细胞高表达音猬因子、神经细胞黏附分子以及神经干细胞标志蛋白巢蛋白和CD133。结论:琼脂糖抗贴壁,神经干细胞培养基诱导培养可使成人BMSCs形成神经球,神经球细胞具有神经干细胞样特征,该方法可为细胞移植修复神经系统损伤提供大量的种子细胞。
OBJECTIVE: To establish an in vitro culture method of inducing adult human bone marrow mesenchymal stem cells (BMSCs) to differentiate into neural stem cells, and to provide seed cells for cell transplantation to repair nervous system injury. Methods: Primary cultured adult BMSCs were cultured and subcultured to the third passage. The cells were inoculated into agar-coated culture flasks and cultured on neural stem cell medium (containing B27, EGF and bFGF). The cultured neurospheres are passaged for amplification. Immunofluorescence staining and Western blotting were used to detect and compare the expression of neural stem cell marker proteins in adherent cultured BMSCs and adherent-cultured neurospheres. RESULTS: Anti-adherent culture induced the formation of neurospheres of BMSCs. The passage of primary neurospheres could be expanded to form secondary neurospheres. Neurosphere cells overexpress sonic hedgehog, neural cell adhesion molecules, and neural stem cell marker nestin and CD133. Conclusion: Agarose-resistant adherent and neural stem cell culture medium can induce adult BMSCs to form neurospheres and neurosphere cells with neural stem cell-like features. This method can provide a large number of seed cells for cell transplantation to repair nervous system damage.