胶质细胞源神经营养因子在大鼠骨髓间充质干细胞神经分化中的表达

Expression of Glial Cell Line-Derived Neurotrophic Factor during Differentiation of Rat Bone Marrow Stromal Cells into Nerve Cells

目的探讨大鼠骨髓间充质干细胞(BMSCs)神经分化及胶质细胞源神经营养因子(GDNF)表达的变化。方法体外培养大鼠BMSCs,传至第4代,行神经诱导分化。流式细胞术检测培养细胞表面的骨髓基质细胞标志CD90和造血细胞标志CD45。以10μg/L碱性成纤维细胞生长因子(bFGF)和(或)10μg/L表皮生长因子(EGF)在含100mL/L胎牛血清(FBS)的DMEM培养基中诱导,7d后观察其细胞形态变化,免疫组织化学法检测其胶质纤维酸性蛋白(GFAP)及神经元特异性烯醇化酶(NSE)的表达,RT-PCR检测其GDNF及其受体RETmRNA的变化。结果BMSCs能在体外成功培养及纯化,第4代细胞高表达CD90(93.4%),不表达CD45。诱导7d后,实验组大鼠均可见GFAP及NSE表达,而对照组为阴性。实验组中bFGF组及bFGF加EGF组表达的NSE高于EGF组,而EGF组表达GFAP更高。RT-PCR检测示BMSCs向神经细胞诱导后,GDNF表达显著增强,并促使RET基因表达,以bFGF加EGF组最明显。结论bFGF及EGF可促进大鼠BMSCs向神经细胞分化,在向神经细胞分化的过程中,能促进GDNF和RET基因的表达,GDNF可能在BMSCs向神经细胞分化过程中起重要作用。

Objective To investigate neural differentiation of rat bone marrow stromal cells（BMSCs） and expression of change of glial cell line-derived neurotrophic factor（GDNF）.Methods BMSCs were harvested from rats and cultured in DMEM supplemented with 100 mL/L fetal bovine serum （FBS）.They were characterized by flow cytometry of CD90 and CD45.At passage 4,BMSCs were induced by basic fibroblast growth factor（bFGF,10 μg/L）,epidermal growth factor（EGF,10 μg/L） and both of them in DMEM with 100 mL/L FBS for 7 days,respectively.The expression of neural specific enolase （NSE） and glial fibrillary acidic protein （GFAP） were detected by immunocytochemistry.The expression of GDNF and RET mRNA were detected by reverse transcription-polymerase chain reaction（RT-PCR）.Results BMSCs at passage 4 were CD90 positive and CD45 negative on flow cytometry.After 7 days of induction,a certain number of cells showed expressions of GFAP and NSE by immunocytochemistry method in experimental group,while the cells showed no expressions of GFAP and NSE in control group.In bFGF group and bFGF plus EGF group,the positive rate of NSE was significantly higher than EGF group,while expression of GFAP in EGF group was obviously higher.BMSCs expressed low level GDNF mRNA and no RET mRNA,however,after induction,GDNF mRNA highly increased and RET mRNA expressed,especially in bFGF plus EGF group.Conclusions bFGF and EGF can not only induce neural differentiation of BMSCs,but also increase the expressions of GDNF and RET significantly.GDNF may play an important role in the process of differentiation of BMSCs into nerve cells.