体外诱导人脐带间充质干细胞向内耳毛细胞样细胞分化的研究

Differentiation of Human Umbilical Cord Mesenchymal Stem Cells into Hair-cell Like Cells in Vitro

目的探讨并建立一种体外定向诱导人脐带间充质干细胞(human umbilical cord mesenchymal stem cells,hUC-MSCs)向内耳毛细胞样细胞分化的方法。方法采用组织贴壁法培养获得hUC-MSCs,流式细胞仪鉴定其表面标记物;神经干细胞诱导阶段分为对照组(DFNB基础诱导培养基)、实验1组(DFNB基础诱导培养基+20ng/ml EGF+20ng/ml bFGF)、实验2组(DFNB基础诱导培养基+20ng/ml EGF+20ng/ml bFGF+琼脂糖包被),培养3~5天后采用免疫荧光法检测各组标记蛋白Nestin的表达;内耳毛细胞样细胞诱导分化阶段,分为对照组(DFNB基础诱导培养基+明胶包被)、诱导组[DFNB基础诱导培养基+20ng/ml EGF+1μM全反式维甲酸/全反式维生素A酸(all trans retinoic aid,ATRA)],培养4周后采用qRT-PCR和免疫荧光法检测各组毛细胞标记物Math1、MyosinⅦa、Brn3c的表达。结果在神经干细胞诱导阶段,对照组、实验1组均无神经球结构,实验2组中hUC-MSCs分化3d后细胞有明显神经球样结构,且Nestin阳性细胞数更高(P<0.05);而向内耳毛细胞样诱导分化4周后,诱导组毛细胞标记物Math1、MyosinⅦa、Brn3c的mRNA和阳性细胞数的表达水平明显升高(P<0.05)。结论体外诱导人脐带间充质干细胞先向神经干细胞方向分化再向内耳毛细胞分化方向诱导,可有效提高毛细胞标记物MyosinⅦa、Brn3c、Math1的表达水平,促进hUC-MSCs向类毛细胞样细胞分化,可能为将来感音神经性聋的内耳移植治疗提供更为适合的种子细胞。

Objective To induce human umbilical cord mesenchymal stem cells（hUC-MSCs）to hair-cell like cells in the inner ear,using a two-step neural differentiation method.Methods The hUC-MSCs were obtained from human umbilical cords by tissue adherence culture,whose surface antigen CD29,CD34,CD44,CD45,CD90,HLA-ABC,and HLA-DR could be identified by flow cytometry.In the neural stem cells induced phase,the NSE positive cells were analyzed by microscope and immunohistochemistry.In the second stage,the expression of hair-cell like cells markers（Math1,MyosinⅦa,Brn3c）were tested by qRT-PCR and immunofluorescence method.Results The control group and the protocol group had little NSE after differentiation while the protocol B group presented a neurobiological structure and demonstrated a higher NSE positive ratio after 5days＇ neural stem cells induction（P0.05）.Compared to the control group,the mRNA and protein level of Math1,MyosinⅦa,and Brn3 cexhibited a significant increase in the differential group,which induced for 4weeks in the hair-cell like cells in the inner ear＇s induced phase（P0.05）.Conclusion The two-stage induction（hUC-MSCs-neural stem cells-hair-cell like cells）could produce more MyosinⅦa,Brn3 cand Math1,which may provide an appropriate way to treat sensorineural deafness.