过表达Nurr1基因的骨髓间充质干细胞的诱导及移植治疗帕金森病的研究

目的:探讨Nurr1基因修饰大鼠骨髓间充质干细胞(mesenchymalstemcells,rMSCs)脑内移植对帕金森病(parkinsondiseasePD)大鼠的治疗作用。方法:用脂质体转染法转染PcDNA3.1(+)-Nurr1入rMSCs并稳定表达.然后移植大鼠PD模型纹状体内;观察行为学变化并用免疫组化、RT-PCR等方法检测移植细胞的Nurr1、DAT和TH表达;利用高效液相色谱检测多巴胺(DA)、二羟苯乙酸(DOPAC)和高香草酸(HVA)含量。结果:Nurr1-rMSCs组和rMSCs组移植治疗8周内PD大鼠旋转行为均得到一定的改善(P<0.05);移植后2～4周Nurr1-rMSCs组较rMSCs组改善程度更为显著(P<0.05),但第8周时二组行为学差异无统计学意义(P>0.05)。免疫组化显示Nurr1-rMSCs能够稳定表达Nurr1且少量细胞表达DAT,但未发现TH阳性细胞。而rMSCs组和对照组则均未发现有Nurr1、DAT、TH表达;RT-PCR检测显示移植后2～8周,Nurr1-rMSCs组移植区有Nurr1和DATmRNA表达,但未发现THmRNA表达;两治疗组DA、DOPAC和HVA含量均较对照组增高(P<0.05)。结论:Nurr1基因转染大鼠骨髓间充质干细胞移植大鼠纹状体可以在一定时期内存活并有效表达,同时可提高纹状体DA含量,改善模型鼠症状,为治疗PD的研究提供了实验依据。

Induction of Nurr1-overexpression mesenchymal stem cells and their transplanation in an animal model of Parkinson disease

Objective:To investigate the therapeutical effect of rat mesenchymal stem cells (rMSCs) transfected with Nurr1 gene on Parkinson disease rat models. Methods:The recombinant pcDNA3.1(+)-Nurr1 were transfected into rMSCs with lipofectamine 2000. rMSCs and Nurr1-gene transfected rMSCs were transplanted into the lesioned striatum of PD models. At different time after transplantation, the rotational behaviors of the rats were observed and the levels of DA and DA metabolites in striatum of the grafted rats ,as well as Nurr1?DAT?TH expression in the striatum of PD rats were also analyzed by using high performance liquid chromatography(HPLC). Results: The rotational behaviors of the rats received grafts of Nurr1-gene modified rMSCs improved apparently 4 weeks after transplantation compared with that of the control group and rMSCs group(P<0.05), however there was no difference between transgenic rMSCs group and rMSCs group at the time of 8 weeks after transplantation(P>0.05). The expression of Nurr1 and DAT were observed during 8 weeks after transplantation according to the results of immunohistochemistry and RT-PCR, but the expression of TH cannot be detected. The levels of DA and DA metabolites in the striatum of the transgenic cells group increased as compared with that of the rMSCs group and control group(P<0.05). Conclusions: rMSCs transfected with Nurr1 gene can survive in the striatum of PD rat model at least 8 weeks after administration, which can express the features of Nurr1 and DAT. This therapeutic method can increase the level of DA and ameliorate the motor ability of PD model. The results provide some reference data of experiment for the clinical trial MSCs and gene therapy in PD.