神经营养因子基因修饰的神经干细胞在帕金森病大鼠模型中的治疗作用

目的 观察人胶质细胞源性神经营养因子(GDNF)转染的大鼠神经于细胞移植对帕金森病(PD)模型大鼠的治疗作用. 方法 制备PD大鼠模型并将成功模型分为PD模型组、神经干细胞组和GDNF基因修饰神经干细胞组.细胞移植后,动态观察动物行为学变化,定量分析黑质多巴胺能神经元、纹状体多巴胺及其代谢产物的变化. 结果 GDNF基因修饰神经干细胞移植能有效改善实验动物的行为学表现,细胞移植后第5周时,PD模型组、神经干细胞组和GDNF基因修饰的神经干细胞组90 min内的旋转圈数分别为(993.9±159.1)圈、(956.7±136.3)圈和(433.6±100.9)圈,GDNF基因修饰的神经干细胞组可显著减少PD模型大鼠向损伤侧旋转的圈数(F=95.694,P=0.000);第7周时,PD模型组的90 min内的旋转圈数为(964.2±152.0)圈,神经干细胞对照组和GDNF基因修饰的神经干细胞组分别为(909.2±136.3)圈和(399.4±84.4)圈(F=106.134,P=0.000);第9周时,PD模型组、神经干细胞组和GDNF基因修饰的神经干细胞组90min内的旋转圈数分别为(909.5±152.2)圈、(865.5±129.1)圈和(312.2±63.7)圈(F=151.100,P=0.000).GDNF基因修饰神经干细胞移植能有效提高损伤侧纹状体内的多巴胺及其代谢产物水平,PD模型组、神经干细胞组和GDNF基因修饰干细胞组毒素注射侧多巴胺含量分别为(3.3±0.3)ng/mg组织、(3.7±1.3)ng/mg组织和(7.5±0.8)ng/mg组织,GDNF基因修饰的干细胞组多巴胺水平明显高于神经干细胞组和PD模型组(F=59.543,P=0.000);GDNF基因修饰神经干细胞组二羟基苯乙酸水平明显高于神经干细胞组和PD模型组,分别为(0.9±0.1)ng/mg组织、(0.6±0.2)ng/mg组织和(0.5±0.1)ng/mg组织(F=17.293,p=0.000);PD模型组、神经干细胞组和GDNF基因修饰干细胞组毒素注射侧高香草酸(HVA)水平分别为(0.5±0.1)ng/mg组织、(0.6±0.2)ng/mg组织和(0.9±0.1)ng/mg组织,GDNF基因修饰神经干细胞组HVA水平明显高于神经干细胞组和PD模型组(F=35.175,P=0.000). 结论 GDNF基因修饰神经干细胞移植能有效改善损伤黑质-纹状体的多巴胺系统功能,GDNF基因治疗具有潜在的临床应用价值.

Glial cell line-derived neurotrophic factor engineered neural stem cell transplantation as a therapeutic approach for Parkinson' s disease in rat model

Objective To explore the therapeutic effects of human glial cell line-derived neurotrophic factor (GDNF)-engineered rat neural stem cell (NSC) transplantation in rat model of Parkinson's disease ( PD) . Methods SD rats received a single injection of 24 μg of 6-hydroxydopamine (6-OHDA) at two sites in right striatum. Then 10 days after surgery, the successful animal models of PD were divided into 3 groups: PD model group ( 2 μl transplantation media was injected in right striatum), NSC group (transplanted were 2×10~5 NSCs infected by bare lentivirus) and GDNF group (transplanted were 2×10~5 GDNF-engineered NSCs). The rotation scores were assessed 5 weeks, 7 weeks and 9 weeks after transplantation. The dopaminergic neurons in substantia nigra ( SN ) were analyzed quantitatively using immunohistochemistry for tyrosine hydroxylase (TH), and the dopamine and its metabolites, dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) were analyzed 9 weeks after transplantation by high performance liquid chromatography ( HPLC) . Results GDNF-engineered NSC transplantation could effectively improve the behavioral performance in rats. At the 5th week after cell transplantation, the rotation turns within 90 min were (993. 9±159. 1) turns, (956. 7±136. 3) turns and (433. 6±100. 9) turns in PD model group, NSC group and GDNF group respectively (F=95. 694, P = 0. 000). At the 7th week, the rotation turns within 90 min were (964. 2 ± 152.0) turns, (909. 2 ± 136. 3) turns and (399. 4±84. 4) turns in PD model group, NSC group and GDNF group respectively (F = 106. 134, P=0. 000). At the 9th week, the rotation turns within 90 min were (909. 5±152. 2) turns, (865. 5± 129. 1) turns and (312. 2±63. 7) turns in PD model group, NSC group and GDNF group respectively (F= 151. 100, P = 0.000). GDNF-engineered NSC transplantation could significantly increase the levels of dopamine and its metabolites in injured striatum. The concentrations of dopamine in injured striatum was higher in GDNF group than that in PD model group and NSC group C(7. 5±0. 8) ng/mg vs. (3.3±0.3) ng/mg and (3. 7±1. 3) ng/mg, F=59. 543, P = 0. 0003. The level of DOPAC was higher in GDNF group than that in PD model group and NSC group C(0. 9±0. 1) ng/mg vs. (0. 5± 0. 1) ng/mg and (0. 6±0. 2) ng/mg, F= 17. 293, P=0. 000]. The concentration of HVA in injured striatum was higher in GDNF group than that in PD model group and NSC group [(0. 9±0. 1) ng/mg vs. (0.5±0. 1) ng/mg and (0. 6±0. 2) ng/mg, F=35.175, P = 0.000]. Conclusions engineered NSC transplantation improves the function of dopamine system in SN and striatum, and GDNF gene therapy has potential clinical value.