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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。
OBJECTIVE: To use bone marrow mesenchymal stem cells (MSCs) as a carrier cell to carry three genes involved in DA synthesis and metabolism: tyrosine hydroxylase (TH), aromatic amino acid decarboxylase (AADC) and triphosphate Guanylate cyclase-I (GCH-I) gene in Parkinson’s disease (PD) model rats. Methods: MSCs were infected with supernatant of recombinant viruses AAV-TH, AAV-AADC and AAVGCH-I in vitro. MSCs carrying TH, AADC and GCH-I genes were transplanted into the striatum of PD model rats, To detect the changes of dopamine and its metabolites in the striatum and substantia nigra and to observe the changes in their behavior to evaluate the therapeutic effect of the above genes on PD rats. Results: MSCs were infected with recombinant pseudovirions and then implanted into PD rats. The expression of these three genes could still be detected 12 weeks after transplantation by reverse transcription polymerase chain reaction (RT-PCR) and in situ hybridization. Immunofluorescence showed that MSCs survived in brain 12 weeks after transplantation. Behavioral observation at 4 weeks, 8 weeks and 12 weeks after transplantation showed that apomorphine (APO) -induced rotational behavior was significantly improved compared with LacZ gene transplanted group <0.01). The triple gene transplants at 12 weeks after transplantation showed a significant improvement over the double gene transplants (p <0.01). Twelve weeks after transplantation, the levels of DA and DOPAC in injured striatum and substantia nigra were determined by high performance liquid chromatography (HPLC). Compared with the triple gene transplantation group hTH + hGCH-I gene transfection group was significantly higher (p <0.01); triple gene transplants than hTH + hAADC gene transplants group increased, but there was no significant difference between the two groups. CONCLUSION: The genetically engineered MSCs expressed the target gene well after transplanted into the brain of PD model rats. In the long-term observation on the improvement of animal behavior and biochemistry, it was found that triple gene combined with intracerebral transplantation may be more effective than the double gene Co-transplantation Better Parkinson’s Disease Gene Therapy Pathway.