neurturin基因转染c17．2神经干细胞移植治疗帕金森病模型大鼠的实验研究

The effect of the neurturin gene-transfected c17. 2 neural stem cells on a rat model of Parkinson disease in an experimental study

目的探讨neurturin（NTN）基因转染c17．2神经干细胞脑内移植后对帕金森病（PD）大鼠模型的治疗作用，并探讨神经干细胞介导的基因治疗方法的有效性。方法在构建成功高表达NTN蛋白NTN-c17．2细胞克隆的基础上，分别在6-羟多巴胺单侧模型大鼠的纹状体区移植入NTN-c17．2、Mock-c17．2和磷酸缓冲液（PBS），通过免疫组化、旋转行为变化和高效液相色谱监测观察c17．2神经干细胞的分化，NTN基因的表达和NTN蛋白的治疗作用。结果NTN-c17．2细胞移植后，标记抗原巢蛋白（nestin）逐渐消失，细胞分化成神经元、星形胶质细胞和少突胶质细胞，但仍持续分泌NTN蛋白。动物模型旋转行为动态观测和高效液相色谱法（HPLC）检测都提示NTN-c17．2和Mock-c17．2好于PBS组，两组之间差异无统计学意义（P＞0．05），RT-PCR和免疫组织化学检测提示c17．2神经干细胞能产生多种神经营养因子，移植后部分细胞可分化成酪氨酸羟化酶（TH）阳性神经细胞。结论用带有NTN基因的神经干细胞移植治疗PD，显示出一定的治疗效果，然而，这种治疗作用主要和神经干细胞分泌的多种神经营养因子和移植后的细胞分化有关，NTN蛋白可以增强这种作用。

Objective To investigate the effect of c17.2 neural stem cells expressing neurturin （NTN） on a rat model of Parkinson disease. Methods The c17.2-NTN clone over-expressing NTN protein was constructed. For three studied groups, NTN-c17. 2, Mock-c17. 2 and phospholate buffer solution （PBS） were injected into the striatum of the 6-hydroxydopamine （6-OHDA）- induced rat model of Parkinson disease. The differentiation of c17.2 neural stem ceils, the expression and neuroregeneration of NTN were detected by immunohistochemical staining, and rotational behavior was estimated by apomorphine injection. The levels of dopamine and its metabolites in the striatum were measured by high performance liquid chromatography （HPLC） with electrochemical detection. Results After intrastriatal grafting NTN-c17. 2, low expression of nestin was gradually observed, while grafts stably overexpressed NTN and differentiated as neurons, astrocytes and oligodendrocytes. Based on regenerative parameters of the asymmetry improvement and the elevated contents of dopamine and its meatbolites, the regenerative effects of NTN-c17.2 group and the Mock-c17.2 group were significantly better than those of PBS group. RT-PCR assays and in vivo histology revealed that NSCs produced neurotrophic factors, and some cells from NTN-c17.2 and Mock-c17.2 cells differentiated to be tyrosine hydroxylase （TH）-positive cells. Conclusions The transplantation of NTN-c17. 2 exerts neuroregenerative effects in rat model of PD. The technic of intracerebral gene mediated by neural stem cells might be a potential method for gene therapy of PD.