GDNF和GM1体外联合诱导骨髓基质干细胞向多巴胺能神经元分化的实验研究

The rat bone marrow stromal cells were induced into dopaminergic neuron by GDNF and GM1 in vitro

目的应用GDNF和GM1体外联合诱导MSCs转化为多巴胺能(DA)神经元。探索体外诱导MSCs定向分化为DA能神经元的最佳条件。方法取雄性Wistar大鼠股骨和胫骨骨髓,密度梯度离心法分离获取单个核细胞。进行MSCs的体外培养和传代扩增。采用贴壁培养法使MSCs得到纯化。依据加入的神经营养因子不同分为对照组及实验组(GM1组、GDNF组、GDNF+GM1组)。诱导过程中在倒置显微镜下观察细胞形态变化,分别在诱导第3天、第7天进行NSE、GFAP、TH免疫细胞化学检测。计数NSE和TH阳性细胞数,并计算阳性细胞百分比。采用SPSS10.0软件进行统计学处理。结果对照组可见少量NSE阳性细胞。各实验组比较发现,GDNF+GM1组NSE阳性细胞率最高,GDNF组次之,GM1组最低。单独应用GDNF和GM1不能诱导MSCs表达TH,联合应用GDNF和GM1可诱导MSCs表达TH。随着诱导时间的延长,TH阳性表达增加。结论GDNF能够单独诱导MSCs向神经元样细胞分化,GM1不能单独诱导MSCs分化为神经元样细胞,但与GDNF联合,不仅可明显促进MSCs向神经元样细胞分化,而且部分细胞能够表达TH。

Objective To approach a way to induce marrow stromal cells（MSCs） to dopaminergic neuron by GDNF and GM1 differentiatly in vitro and supply a ideal cells source for treating Parkinson＇s disease. Methods The rat MSCs were isolated primarily from the femurs and tibias of the Wistar rats. MSCs were cultured ,proliferated and purified by passage culture. Cultuered MSCs were divided into control and experimental group （GM1 group,GDNF group,GDNF＋GM1group）. The surface markers of the differentiated neuron,such as NSE and TH were detected by immunocytochemistry after MSCs were cultured in induction media for 3 days and 7 days. Results In control groups ,the NSE expression of MSCs was very low,expression of NSE increased in two experimental group excepting GM1 group. There were no TH positive cells when MSCs were incubated in induction media only containing GDNF or GM1. A few of TH-positive cells began to appear after being cultured in media containing GDNF and GM1. The percentage of TH-positive cells on 7d was increased more significantly than that on 3d. Conclusion In vitro,GDNF could induce MSCs to differentiate into neuron-like cells. GM1 could not induce MSCs to differentiate into neuron,but it can promote MSCs to differentiate into neuron with GDNF and induce MSCs into dopaminergic neuron which expressed TH.