体外诱导牙髓干细胞向神经元细胞分化方法的初步研究

摘要

目的:体外探究牙髓干细胞(DPSC)向神经元分化的诱导方法.方法:先利用神经干细胞(NSC)体外诱导培养液诱导DPSC分化为NSC样细胞,再用神经元体外诱导培养液诱导已分化为NSC样细胞,使其进一步分化为神经元样细胞.DPSC向神经元分化的过程中用光学显微镜观察细胞形态;用荧光实时定量PCR、免疫荧光染色检测不同阶段神经表面标记物(Nestin、βⅢ-tubulin、NF-L)基因和蛋白的表达变化.结果:在NSC体外诱导培养液的作用下,DPSC能分化为类似于NSC样细胞形态,并呈现神经球样聚团生长;在神经元体外诱导培养液的作用下,该神经球能进一步分化为类似于神经元样细胞形态;DPSC向神经元细胞分化的过程中,与分化0d时相比,神经表面标记物Nestin、βⅢ-tubulin、NF-L基因的表达水平明显升高(P＜0.05).结论:DPSC有分化为成熟的神经元样细胞的潜能.%AIM:To develop an in vitro protocol for the induction of human dental pulp stem cells (DP-SCs) into neuronal-like cells.METHODS:DPSCs were induced by neural stem cells (NSCs) conditioned medium for 10 days to differentiate into NSCs-like cells.Then NSCs-like cells were induced by neurobasal medium supplemented with neurotrophic factors for 4 days to differentiate into neuronal-like cells.The morphology of the cells was observed by microscope.The gene and protein expression of neuronal specific markers (nestin,β Ⅲ-tubulin,NF-L)were detected by quantitative Real-time PCR and immunocytochemistry,respectively.RESULTS:In the initial step,the attached fibroblast-like DPSCs was changed into the floating neurosphere-like bodies on low-attachment culture surfaces.With the addition of neurotrophic factors to the neurobasal medium,the NSCs-like cells from DPSCs demonstrated a more complex neuronal morphology change.The gene and protein expression of nestin,β Ⅲ-tubulin and NF-L were increased over time in culture.CONCLUSION:DPSCs can be induced to differentiate into the neuronal-like cells.