大鼠骨髓间充质干细胞体外向肠神经细胞分化及胶质细胞源神经营养因子表达的变化

Differentiation of rat bone marrow stromal cells into enteric neurons and expression change of glial cell line-derived neurotrophic factor

目的 探讨大鼠骨髓间充质干细胞向肠神经分化的条件及胶质细胞源神经营养因子（glial cellline-derived neurotrophic factor,GDNF）及其受体RET基因表达的变化.方法 体外培养大鼠骨髓间充质干细胞（bone marrow stromal cells,BMSCs）,传至第四代,进行诱导分化.以10ng/ml GDNF和10%FBS的稀释胎肠培养基（fetal gut culture medium,FCCM）以及仅含有10 ng/ml GDNF的L-DMEM诱导7 d,对照组为含10%FBS的L-DMEM完全培养液,免疫组化的方法鉴定细胞的神经特异性烯醇化酶（neural specific enolase,NSE）、血管活性肠肽（vasoactive intestinal peptide,VIP）及一氧化氮合酶（nit ric oxide synthase,nNOS）的表达.RT-PCR检测GDNF及RET mRNA的变化,Westem Blot方法检测GDNF蛋白的表达情况.结果 BMSCs能在体外成功培养及纯化,诱导7 d后,实验组均可见NSE、VIP及nNOS的表达,两实验组的阳性率有统计学差异,而对照组为阴性.RT-PCR检测示,BMSCs向肠神经细胞诱导后,GDNF表达显著增强,并促使RET基因表达Western Blot结果提示,诱导后的细胞GDNF在蛋白水平上表达增强.结论 GDNF联合FCCM可诱导BMSCs分化为肠神经细胞,在向神经细胞分化的过程中,能促进GDNF表达的增强,并促使RET基因的表达,GDNF-RET信号通路在肠神经分化过程中可能发挥着重要作用.

Objective To investigate differentiation conditions of bone marrow stromal cells （BMSCs） to enteric neuron in vitro and expression of glial cell line-derived neurotrophic factor （GDNF） and its receptor RET. Methods BMSCs were harvested from rats and cultured in DMEM supplemented with 10% fetal bovine serum （FBS）. After 4 passages, BMSCs were induced with 10ng/mlGDNF and fetal gut culture medium （FGCM） for 7 days. The expression of neural specific enolase （NSE）. vasoactive intestinal peptide （VIP） and nitric oxide synthase （nNOS） was examined with immunohistochemistry. The change of GDNF and RET mRNA expressed was assessed with RT-PCR and protein expression was quantified with western blot. Results BMSCs were cultured and purified in vitro. After 7 days of induction, a certain number of cells showed expression of NSE, VIP and nNOS by immunohistochemistry method in experimental group, while the cells showed no expression of VIP and NSE in control group. BMSC expressed low GDNF and no RET mRNA, while after induction, the expression of GDNF mRNA was highly increased and RET mRNA expressed. GDNF protein was expressed. Conclusions GDNF and FGCM induce differentiation of BMSCs into enteric neurons,and GDNF-RET signaling pathway may play an important role in enteric neural differentiation.