Nurr1基因转染人脐血间充质干细胞诱导分化为多巴胺能神经元的实验研究

Differentiation of human umbilical cord blood mesenchymal stem cells transfected by Nurr1 gene into dopaminergic neurons in vitro

目的探讨外源性Nurr1基因修饰的人脐血间充质干细胞在基因重组成纤维细胞生长因子-8（FGF8）和音猬因子（Shh）诱导下体外分化为多巴胺能神经元的情况。方法间充质干细胞被随机分为A组（对照组）、B组（Nurr1组）、C组（FGF8＋Shh组）及D组（Nurr1＋FGF8＋Shh），采用脂质体法将pcDNA3．1-Nurr1转染B、D组间充质干细胞，Western blot观察Nurr1基因表达情况，并在神经元条件培养液中进行增殖培养和诱导分化，间接免疫荧光染色法鉴定细胞性质，高效液相色谱法测定多巴胺含量。结果Western blot结果显示转染后Nurr1蛋白表达明显增高。A组和B组未发现酪氨酸羟化酶（TH）阳性细胞，而C组和D组TH阳性细胞分别为10．12％±2．65％及25．36％±3．13％，多巴胺含量分别为（43．6±2．1）nmol／L及（83．2＋3．5）nmol／L，差异均有显著性意义（P〈0．01）。结论人脐血间充质干细胞在FGF8和Shh诱导下可以分化为多巴胺能神经元，外源性Nurr1基因修饰后．分化为多巴胺能神经元的数量增加。

Objective To investigate the differentiation of human umbilical cord blood mesenchymal stem cells （hUMSCs） transfected by exogenous Nurr1 gene into dopaminergic neurons in vitro by fibroblast growth factor 8 （FGFS） and sonic hedgehog （Shh）. Methods hUMSCs were divided randomly into group A （control group）, group B （Nurr1 group）, group C （FGF8 ＋Shh group） and group D （Nurr1＋FGF8＋Shh group）, and induced in the neuronal conditioned medium （NCM）. Group B and D hUMSCs were transfected with pcDNA3.1-Nurr1 plasmid by lipofectamine and selected by G418. The expression ofNurr1 gene in hUMSCs was detected by Western blot. The cell type was identified by redirect immunofluorescence staining. The concentration of dopamine was analyzed by high performance liquid chromatography. Results Western blot showed that Nurr1 gene was over-expressed after transfection. No TH-positive cells were found in group A and B. The TH-positive rates of group C and D were 10.12%±2.65% and 25.36%±3.13% （P〈0.01）, and the concentrations of dopamine were （43.6±2.1） and （83.2±3.5） nmol/L （P〈0.01）, respectively. Conclusion hUMSCs can differentiate into dopaminergic neurons by FGF8 and Shh, and the number of dopaminergic neuron would increase after transfection of exogenous Nurr1 gene.