RA、SHH和bFGF体外诱导人牙髓干细胞向神经细胞分化的研究  

Effects of RA,SHH and bFGF to induce human dental pulp stem cells into neuron in vitro

作　　者：张智慧[1] 胡伟平[1] 郭阳[1] 曹潇方[1] 

机构地区：[1]哈尔滨医科大学附属第二医院口腔修复科,黑龙江哈尔滨150086

出　　处：《牙体牙髓牙周病学杂志》2011年第2期72-76,共5页Chinese Journal of Conservative Dentistry

基　　金：黑龙江省自然科学基金资助项目(D200856)

摘　　要：目的:研究体外使用全反式维甲酸(all-trans-retinoic acid,RA)、音猬因子(sonic hedgehog,SHH)、碱性成纤维生长因子(fibroblastgrowth factor-basic,bFGF)体外诱导人牙髓干细胞(dental pulp stem cells,DPSCs)分化为神经细胞的可行性,以优化DPSCs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DPSCs,并进行克隆化培养,检测STRO-1的表达。将DPSCs分别接种于含有不同浓度诱导液,MTT法检测三种因子对细胞增殖能力的影响,免疫荧光法检测微管相关蛋白(microtubule-associated protein-2,MAP-2)、神经元烯醇化酶(neuron-specific enolase,NSE)、胶质原纤维酸性蛋白(glial fibrillary acid protein,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STRO-1表达阳性。三种因子联合诱导促增殖作用最强,与其他组比较,差异有统计学意义(P<0.05)。除对照组外,各诱导组均检测出神经元样细胞,其中以三因子联合诱导组阳性细胞表达率最高,促分化作用均优于其他组。透射电镜观察到神经元样细胞表现。结论:三因子联合可以在体外有效诱导人DPSCs转化为神经细胞。AIM： To examine the inducing effects of all-trans-retinoic acid（RA）,sonic hedgehog（SHH） and fibroblastgrowth factor-basic（bFGF） from human dental pulp stem cells into neurons.METHODS： Human dental pulp was extracted from the first premolars or impacted third molars.A single cell suspension was acquaired by enzymatic digestion and filtration.A limited dilution method was used to culture the isolated primary DPSCs,which was expanded by cloning training.DPSCs in passage 3 were subjected to different inducing medium containing bFGF＋RA,bFGF＋SHH and bFGF＋RA＋SHH respectively.The proliferation of the induced DPSCs was measured by MTT method.Expression of microtubule-associated protein-2,neuronspecific enolase and glial fibrillary acidic protein was detected by immune fluorescent assay.The ultrastructure of the cells were examined by transmission electron microscopy（TEM）.RESULTS： Positive expression of STRO-1 was detected in clone-derived cells.The inducing medium containing RA＋SHH＋bFGF showed the strongest proliferation capacity,with a significantly higher proliferation rate as compared with other groups （P0.05）.Neuron-like cells were found in all three groups,with most cell number in the RA＋SHH＋bFGF group.CONCLUSION： The conbination of RA,SHH and bFGF could effectively induce DPSCs into neural-like and /or glia-like cells in vitro.

关 键 词：人牙髓干细胞 全反式维甲酸 音猬因子 碱性成纤维生长因子 分化 

分 类 号：R780.2[医药卫生—口腔医学]