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目的建立一种可同时高效稳定表达酪氨酸羟化酶(TH)和胶质细胞源性神经营养因子(GDNF)的双基因工程细胞,探讨其在帕金森病(PD)基因治疗中的作用。方法应用分子克隆技术构建真核表达载体PcDNA3.1/hGDNF和PcDNA3.0/hTH,并同时转染至SHSY5Y细胞系,利用半定量逆转录聚合酶链反应(RTPCR)鉴定筛选出的阳性细胞克隆TH和GDNF的表达水平,将双转工程细胞与MN9D细胞共培养,高效液相色谱分析工程细胞对MN9D细胞生长状态及细胞内单胺类递质含量的影响。结果双转基因工程细胞可防止MN9D细胞退变死亡,与对照组相比较,DA、HVA和DOPAC提高了1.9、2.1和1.7倍(P<0.01)。双转基因工程细胞可对抗MPP+对MN9D的毒性损伤作用,与对照组相比较,DA、HVA和DOPAC提高了3.3、1.7和2.0倍(P<0.01),而且其效果明显优于TH单基因工程细胞。结论成功构建可分泌人TH和GDNF的双转基因工程细胞,其对多巴胺能神经元具有明显的保护作用,其在PD基因治疗中可能具有防治兼顾作用
OBJECTIVE: To establish a double genetically engineered cell line capable of expressing both tyrosine hydroxylase (TH) and glial cell line-derived neurotrophic factor (GDNF) efficiently and stably and to explore its role in the gene therapy of Parkinson’s disease . Methods The eukaryotic expression vectors pcDNA3.1 / hGDNF and pcDNA3.0 / hTH were constructed by molecular cloning technique and transfected into SHSY5Y cell line at the same time. The positive clones screened by semi-quantitative reverse transcriptase-polymerase chain reaction (RTPCR) TH and GDNF expression levels of co-cultured double-engineered cells and MN9D cells, high performance liquid chromatography analysis of engineering cells on MN9D cell growth status and intracellular monoamine neurotransmitters content. Results Double transgenic mice could prevent the degeneration of MN9D cells. Compared with the control group, DA, HVA and DOPAC increased 1.9, 2.1 and 1.7 times (P <0.01), respectively. Double transgenic cells could antagonize the toxic effects of MPP + on MN9D cells. Compared with the control group, DA, HVA and DOPAC increased by 3.3, 1.7 and 2.0 times (P <0.01), and the effect was obviously better than TH single genetically engineered cells . Conclusion The double-transgenic engineering cells that can secrete human TH and GDNF were successfully constructed, which have obvious protective effect on dopaminergic neurons. It may have both prevention and treatment in PD gene therapy