人牙周膜干细胞体外诱导分化为神经元样细胞的实验研究.doc

人牙周膜干细胞体外诱导分化为神经元样细胞的实验研究 人牙周膜干细胞体外诱导分化为神经元样细胞的实验研究 中作者：甄蕾 刘宏伟 作者单位：同济大学口腔医院 口腔内科，上海 200072 【摘要】 　目的 体外定向诱导人牙周膜干细胞分化为神经元样细胞，探讨人牙周膜干细胞的多向分化潜能。方法 体外分离、培养人牙周膜细胞，待细胞达一定量后用有限稀释法进行克隆化培养，筛选鉴定牙周膜干细胞（PDLSC），用含10 Μg/L碱性成纤维细胞生长因子（bFGF）的预诱导培养液诱导培养24 h，然后换用含5 mmol/L Β-巯基乙醇（Β-ME）的诱导培养液诱导培养6 h，光镜下观察诱导细胞形态学的改变，免疫荧光、RT-PCR等方法检测鼠抗人神经元特异性烯醇化酶（NSE）、神经丝蛋白（NF）和胶质纤维酸性蛋白（GFAP）的表达。同时以未诱导细胞为对照。结果 体外诱导培养6 h细胞即发生形态学改变，可看到典型的神经元样细胞；免疫荧光、RT-PCR检测诱导细胞表达神经元细胞特异性标志NSE、NF，未诱导细胞无表达。结论 人牙周膜干细胞在体外可以诱导分化为神经元样细胞，具有多向分化的潜能。 【关键词】 牙周膜干细胞 神经元样细胞 诱导分化 　　Differentiation of human periodontal ligament stem cells into neuron-like cells in vitro ZHEN Lei, LIU Hong-wei. （Dept. of Oral Medicine, Hospital of Stomatology, Tongji University, Shanghai 200072, China） 　　[Abstract] Objective To isolate and purify the human periodontal ligament stem cells（PDLSC） and investigatethe differentiation potentials of PDLSC into neuron-like cells in vitro. Methods PDLSC were isolated and cultivated.PDLSC of passage 2 was plated at a density of 5×103 per mL. At 80% confluence, the PDLSC were preinduced for 24hours, and were subsequently replaced with an inducing medium containing certain concentration of Β-mercaptoethanal（Β-ME）. After 6 hours of induction, the results were evaluated by morphological observation, immunocytochemicalstaining for neuron specific enolase（NSE）, neurofilament（NF） and glial fibrillary acid protein（GFAP） expression and RT-PCR for NSE, NF, GFAP mRNA. Meanwhile, the uninduced PDLSC were used as a negative control. Results PDLSC could be differentiate into cells with typical neuronal morphology. Immunohisto-chemistry and RT-PCR con-firmed that the induced cells expressed NSE and NF, two marked enzymes of neuron cell. Conclusion PDLSC can be induced into neuron-like cells in vitro. PDLSC have the capability of multilineage differentiations. 　　[Key words] periodontal ligament stem cells； neuron-like cells； induced differentiation 　　牙周膜细胞是牙周组织的主要功能细胞，在牙周组织再生中起重要作用[1]。大量