SHH和bFGF体外诱导人牙髓干细胞向神经细胞分化的研究

目的：研究体外使用音猬因子（SHH）、碱性成纤维生长因子（bFGF）体外诱导人牙髓干细胞（DPSCs）分化为神经细胞的可行性,以优化人牙髓干细胞向神经细胞分化的诱导条件。方法：从因正畸或阻生拔除的第一前磨牙或第三磨牙中提取牙髓,采用酶消化及过滤法得到单细胞悬液,有限稀释法培养分离的原代人牙髓干细胞,并进行克隆化培养,检测间充质干细胞特异性标志物STRO-1的表达。将人牙髓干细胞分别接种于含有不同浓度诱导液,MTT法检测不同时间、两种因子单独或联合对细胞增殖能力的影;免疫荧光法检测抗微管相关蛋白（MAP-2）、神经元烯醇化酶（NSE）、胶质原纤维酸性蛋白（GFAP）的表达。透射电镜观察诱导前后细胞超微结构。结果：克隆来源细胞的间充质干细胞特异性标志物STRO-1表达阳性。100μg/L音猬因子SHH与20μg/L碱性成纤维生长因子bFGF单独作用促增殖作用最强（P〈0.05）,碱性成纤维生长因子bFGF单独作用各组及对照组均未检测出神经元样细胞。音猬因子SHH作用各组检测到阳性细胞。而100μg/L音猬因子SHH与20μg/L碱性成纤维生长因子bFGF联合增殖和分化作用均优于其它组。透射电镜观察到神经元样细胞表现。结论：100μg/L音猬因子和20μg/L碱性成纤维生长因子联合可以在体外有效诱导人牙髓干细胞分化为神经细胞。

Research of SHH and bFGF Inducing Human Dental Pulp Stem Cells into Neuron in Vitro

Objective：To optimize the differentiation conditions of human dental pulp stem cells,and the feasibility of SHH and bFGF inducing human dental pulp stem cells（DPSCs）into neuron in vitro.Methods：The pulp was extracted from the first premolars or the third molars extracted for orthodontics.A single cell suspension was acquired by enzymatic digestion and filtration.A limited dilution method was used to culture primary DPSCs isolated,which was expanded by cloning training and detected the expression of mesenchymal stem cell-specific markers（STRO-1）.MTT was used to assay the changing of the proliferation ability of DPSCs,which were inoculated in different concentrations of the two factors alone or in combination at different times.The immune fluorescent was used in identifing the expression of the microtubule-associated protein-2,neuron-specific enolase and glial fibrillary acidic protein,respectively.And the ultrastructure of human dental pulp stem cells and human dental pulp stem cells induced were detect by the transmission electron microscopy（TEM）.Results：The expression of mesenchymal stem cell-specific markers of clone-derived cells was positive.100μg/L sonic hedgehog or 20μg/L fibroblast growth factor-basic alone showed the strongest capacity of proliferation（P〈0.05）.The neuron-like cells were not detected in each group of bFGF and the control group.Positive cells were detected in each group of SHH.The group of 100μg/L sonic hedgehog and 20μg/L fibroblast growth factor-basic combined was better than others in the role of proliferation and differentiation.Conclusion：100μg/L SHH and 20μg/L bFGF combined could effectively induce DPSCs into neural-like and/or glia-like cells in vitro.