人脐带间充质干细胞源的多巴胺分泌细胞治疗帕金森病模型大鼠的研究  

Study of dopamine-producing cells derived from human umbilical cord mesenchymal stem cells in treatment in rats with Parkinson's disease

作　　者：谢姜[1,2] 王斌 董方 张宏炜 张力伟 白钢[2] 刘拥军 

机构地区：[1]天津市干细胞再生医学转化企业重点实验室天津和泽干细胞科技有限公司博士后科研工作站,300304 [2]南开大学药学院南开大学博士后流动站

出　　处：《天津医药》2017年第1期21-24,I0002,共5页Tianjin Medical Journal

基　　金：天津市科技计划项目(14TXSYJC00468;14ZXCXSY00125)

摘　　要：目的探讨人脐带间充质干细胞(h UC-MSCs)源的多巴胺分泌细胞(h UC-MSCs-DPCs)对帕金森病(PD)模型大鼠的疗效及机制。方法体外培养并诱导h UC-MSCs分化为DPCs,采用ELISA法通过检测多巴胺(DA)、胶质细胞源性神经营养因子(GDNF)表达水平对诱导后的DPCs进行鉴定。SD大鼠大脑右侧黑质区注射6-羟基多巴胺(6-OHDA)制备PD模型。60只PD模型鼠随机分为h UC-MSCs组(移植5×105个h UC-MSCs至纹状体,20只)、h UC-MSCs-DPCs组(移植5×105个h UC-MSCs-DPCs,20只)、对照组[注射等体积磷酸盐缓冲液(PBS),20只)],采用CM-Dil标记移植细胞。移植后4、8和12周,皮下注射阿扑吗啡,观察其诱导的旋转行为;ELISA法检测3组GDNF表达情况。移植后12周处死大鼠,制备脑冰冻切片,免疫荧光染色观察酪氨酸羟化酶(TH)表达情况,FLUORO-JADE?C染色观察神经元生长情况。结果 h UC-MSCs-DPCs诱导成功并且高表达DA和GDNF。移植后4、8、12周,相比对照组和h UC-MSCs组,h UC-MSCs-DPCs组大鼠旋转行为明显改善,GDNF表达量明显升高(P<0.05)。移植后12周,h UC-MSCs-DPCs组大鼠毁损侧纹状体以及双侧黑质区可见TH+细胞,变性神经元数目较对照组明显减少(P<0.05)。结论 h UC-MSCs-DPCs可改善阿扑吗啡诱发的PD模型大鼠的旋转行为,这可能与提高毁损侧纹状体内DA、GDNF表达水平,保护神经元有关。Objective To investigate the efficacy of dopamine producing cells（DPCs） derived from human umbilicalcord-derived mesenchymal stem cells（h UC-MSCs） in treatment in model rats with Parkinson’s disease（PD）. Methods The cultured h UC-MSCs were induced into DPCs in vitro. The dopamine（DA） and glial cell line-derived neurotrophic factor（GDNF） expression levels were detected by ELISA to identify the DPCs. The PD rat model was established by injecting 6-OHDA into the right substantia nigra（SN）. A total of 60 successfulled PD model rats were randomized into h UC-MSCs-DPCs group（5×105h UC-MSCs-DPCs were transplanted into right striatum, n=20）, h UC-MSCs group（5×105h UC-MSCswere transplanted, n=20） and control group（same volume PBS, n=20）. All the transplanted cells were labeled with CM-Dil.The apomorphine induced rotation behavior was assessed at 4, 8 and 12 weeks after cell transplantation. The rats wereexecuted after 12 weeks. The immunofluorescence staining was used to detect the tyrosine hydroxylase（TH） expression level,and FLUORO-JADE？C staining was used to test the apoptotic neurons in brain of rats. Results The h UC-MSCs-DPCswere induced successfully in vitro, which showed a high expression level of DA and GDNF. Furthermore, at 4, 8 and 12weeks after cell transplantation, the rotation behavior was improved, and expression levels of GDNF were significantly higherin h UC-MSCs-DPCs group than those of h UC-MSCs group and control group（P〈0.05）. In addition, we found that most ofthe transplanted TH＋h UC-MSCs-DPCs at the right striatum and a few cells around both the left and the right substantianigra at 12 weeks after transplantation. The apoptotic neurons were decreased after cell transplantation in h UC-MSCs-DPCsgroup than that of control group（P〈0.05）. Conclusion The h UC- MSCs- DPCs can improve the rotational behaviorinduced by apomorphine in PD model rats, which may be involved in improving levels of DA and GDNF in damaged striatumand protecting neurons.

关 键 词：间质干细胞 脐血干细胞移植 帕金森病 多巴胺 纹状体黑质变性 大鼠 Sprague-Dawley 胶质细胞源性神经营养因子 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学] R742[医药卫生—基础医学]