牙髓干细胞向神经细胞方向的诱导分化实验

Experiment on inducing human dental pulp stem cells into neural-like cells

目的探讨克隆化培养分离的人牙髓干细胞是否具有向神经细胞方向分化的潜能,并确定其诱导条件。方法克隆化培养的人牙髓干细胞预诱导24h,然后换含一定浓度二甲基亚砜(DMSO)、丁羟基茴香醚(BHA)、forskolin、β-巯基乙醇(β-ME)和氢化可的松(hydrocortisone)的联合诱导液连续诱导4d,对诱导细胞进行形态学观察,神经胶质酸性蛋白(GFAP)、非特异性酯酶(NSE)免疫组化染色和GFAP mRNA RT-PCR检测。同时,以未诱导细胞为对照。结果诱导12h时细胞形态开始改变,24h时分化为较为典型的神经细胞样细胞,继续诱导分化细胞数量增多;诱导细胞表达神经元细胞特异性标志NSE和GFAP蛋白;RT-PCR检测诱导细胞表达GFAP mRNA,而未诱导细胞均无上述改变和表达。结论人牙髓干细胞在一定的诱导条件下可横向分化为神经元样细胞。

Objective To explore the multi-differentiated capability of human dental pulp stem cells （hDPSCs） obtained by cell-clone culture approach and to determine the appropriate induced medium. Methods The cloned isolation and expansion of hDPSCs were preinduced for 24 h, and were subsequently replaced with neural-inductive medium containing certain concentration of dimethylsulfoxide （DMSO）, butylated hydroxyanisode （BHA）, forskohn, β- mercaptoethanol （β-ME） and hydrocortisone for 4 days. Then induced cells were analyzed by morphological observation, immnocytochemical staining for non-specific esterase （NSE） and glial fibrillary acidic protein （GFAP） expression, RT-PCR for GFAP mRNA. Meanwhile, the uninduced hDPSCs were used as negative control. Results The morphol- ogy of induced cells changed at the initial 12 b, and displayed a typical neuron-like cells at 24 h. There was a gradual increase in the number of these neuronal differentiated cells with continuous induction. Furthermore, immnocytochemical staining showed that the induced cell expressed NSE and GFAP, two marked enzymes of neuron cell. The GFAP mRNA was also detected in induced ceils by RT-PCR assay. In contrast, the uninduced ceils maintained its original appearance and had no expression on them. Conclusion hDPSCs may possess potential of muhiple-differentiation and may differentiate into neuron-like cells on certain inductive condition.