多巴胺合成相关酶基因联合治疗帕金森病大鼠模型的研究

Therapeutic Benefit of TH,AADC,and GCH-I Genes for Parkinson's Disease in Rat Model

目的:利用骨髓间充质干细胞(MSCs)作为运载细胞,携带DA合成代谢过程中3个重要相关酶的基因:酪氨酸羟化酶(TH)、芳香族氨基酸脱羧酶(AADC)和三磷酸鸟苷酸环化水解酶-I(GCH-I)基因,治疗帕金森病(PD)模型大鼠。方法:首先用重组病毒AAV-TH、AAV-AADC和AAVGCH-I的上清液对MSCs进行体外感染;将携带有TH、AADC和GCH-I基因的MSCs移植到PD模型大鼠纹状体内,检测纹状体及黑质内多巴胺及其代谢产物的变化,并观察其行为学的变化,以评估上述基因对PD大鼠的治疗作用。结果:重组假病毒颗粒感染MSCs后植入PD模型大鼠损伤侧纹状体内。通过逆转录聚合酶链反应(RT-PCR)和原位杂交的方法在移植后12周仍能检测到上述3种基因的表达。免疫荧光检测发现移植后12周MSCs在脑内存活良好;移植后4周、8周、12周行为学观察发现阿扑吗啡(APO)诱导旋转行为较对照组LacZ基因移植组有明显改善(p<0.01);移植后12w时三重基因移植组较双重基因移植组有明显改善(p<0.01)。移植后12周用高效液相色谱(HPLC)电化学法测定损伤侧纹状体和黑质内DA及其代谢产物3,4-二羟苯丙酸(DOPAC)的含量,三重基因移植组较hTH+hGCH-I基因移植组有明显增高(p<0.01);三重基因移植组较hTH+hAADC基因移植组有所增高,但两组之间没有显著性差异。结论:基因工程改造的MSCs在移植到PD模型大鼠脑内后可以很好地表达目的基因并在对动物行为学及生化方面改善的长期观察中发现三重基因联合脑内移植可能是比双重基因联合移植更佳的帕金森病基因治疗途径。

Parkinson＇ s disease （PD） is a common neurodegenerative disorder with no effective protective treatment, characterized by a massive degeneration of dopaminergic neurons in the substantia nigra （SNpc） and the subsequent loss of their projecting nerve fibers in the striatum. The major neurochemical manifestation of this disorder is the loss of the neurotransmitter dopamine （DA） in the striatum as a result of the progressive degeneration of the dopaminergic neurons in the substantia nigra. There have been significant progresses in recent years reporting on the use of mesenchymal stem cells （MSCs） in gene therapy, with specific application towards PD. MSCs, a kind of multipotent adult progenitor cells, are considered as a useful vehicle for cell and gene therapy because of their muhiple differentiation potentiality and self-transplantation. The present study was focused on treating rat model of PD using human tyrosine hydroxylase gene （hTH） , human aromatic L-amino acid decarboxylase gene （hAADC） and human GT Pcyclohydrolase I gene （hGCH-I） engineered MSCs, in order to provide a better understanding about the application of these cells in the therapeutic hTH,hAADC and hGCH-I were introduced via recombinant adeno-associated virus benifit of PD. The gene of （rAAV） infection into the MSCs in vitro. The genetically modified MSCs expressing hTH, hAADC and hGCH-I were transplanted into the striatum of PD rat models. The behavior, the nigra-striatal level of DA and its metabolite were detected. The results of present study were shown as follows ： hTH, hAAI）C, hGCH-I and LacZ gene were transfected into MSCs with adeno-associated virus vectors. The HEK293 packaging cells （ATCC） were transfected with the plasmids of pAAV-hTH, pAAV-hAADC, pAAV-hGCH-I, pAAV-LacZ, pAAV-RC, pHelper by using calcium phosphate precipitation. Titer was detected using HT1080 cells. Viral particles were collected and used to infect MSCs. The purified modified MSCs expressing the three kinds of genes were selected separately and were grafted in the striatum of the PD model rats in the lesion side. The MSCs genetically modified suvived well 12 weeks after transplantation. The improvements of the behavior were observed every week after transplantation. Compared with the control group, the rounds of asymmetric rotation after apomorphine administration decreased in the groups double or triple genes engineered MSCs grafted（p 〈 0.01 ）. During the last 4 weeks, the behavioral recovery of triple gene transplantation group was much better than the double gene transplantation groups （ p 〈 0.01 ）. The levels of dopamine and its metabolites in the striatum and substantia nigra were measured 12 weeks after transplantation using HPLC. The productions of DA and its metabolite DOPAC in double or triple gene transplantation groups remarkably increased comparing with control group （ p 〈 0.01 ）. There are significant differences between hTH ＋ hGCH-I and triple gene transplantation groups （ p 〈 0.01 ）, however there are no significant differences between hTH ＋ hAADC and triple gene transplantation groups. The mRNA of hTH, hAADC and hGCH-I in the tissues from transplantation area of rat brains using RT-PCR and the mRNA of the same genes using in situ hybridization 12 weeks after transplantation were detected. Taken together, the present study had successfully introduced the target genes hTH, hAADC and hGCH-I into the striatum of rat PD models via cell vehicle of MSCs. And the three trarget genes can be well expressed by the host rats. Nevertheless, the hTH, hAADC and hGCH-I genes together functionally improved the behavior of PD rats by increasing the levels of dopamine and its metabolites in the striatum and substantia nigra. All the results suggested that the transplantation of MSCs with triple genes might more effective than those expressing double genes.