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| 人PINK1-shRNA载体的构建及对神经母细胞瘤细胞线粒体的影响* |
| Construction of Human PINK1 shRNA Plasmid Vector and its Effect on Mitochondrial Morphology of Human Neuroblastoma SH-SY5Y Cells |
| 投稿时间：2020-04-20 修订日期：2020-05-07 |
| DOI： |
| 中文关键词: PINK1基因shRNA质粒、SH-SY5Y细胞、线粒体功能障碍、帕金森病 |
| 英文关键词: PINK1 gene shRNA plasmid, SH-SY5Y cells, mitochondrial dysfunction, Parkinson |
| 基金项目:北京中医药大学校级自主课题(2019-XJ-SYJJ-010；2015-JYB-JSMS019) |
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| 中文摘要: |
| 目的:构建筛选人源靶向特异PINK1-shRNA敲减质粒,转染神经母细胞瘤SH-SY5Y细胞并验证该质粒转染后对PINK1基因的敲减效率,观察对细胞线粒体形态的影响,为进一步探讨PINK1基因在帕金森病的发病机制中可能具有的作用提供细胞模型和实验基础。方法: 构建两对人源PINK1-shRNA序列(编号分别为PINK1-shRNA-39和PINK1-shRNA-42),将这2对干扰序列连接在载体上形成重组载体,经测序验证后,将空载体和两对敲减质粒分别转染SH-SY5Y细胞,获得基因敲减的细胞模型,用CCK-8法检测细胞的存活率,用荧光定量PCR方法确定PINK1基因的敲减效率,用蛋白质免疫印迹法验证细胞内PINK1的表达水平是否发生改变,用激光共聚焦显微镜观察线粒体的形态是否发生了改变。结果: 我们提取的质粒,经测序结果显示,质粒载体构建成功；转染细胞后,CCK8法检测细胞存活率发生了降低,与正常组比较,PINK1-shRNA-39和PINK1-shRNA-42敲减质粒组,细胞的存活率分别降低了13.7%(P＜0.05)和14.1%(P＜0.05)；荧光定量PCR结果显示,与正常组相比,PINK1-shRNA-39和PINK1-shRNA-42敲减质粒转染的细胞内PINK1基因的表达分别降低了24.1%(P＜0.01)和36.7%(P＜0.01)；蛋白质印迹法结果显示,与正常组相比,两对质粒分别转染细胞后,PINK1-shRNA-39和PINK1-shRNA-42敲减质粒转染的细胞内PINK1蛋白的表达水平降低,差异有统计学意义(P＜0.01),PINK1-shRNA-42敲减质粒转染的细胞内PINK1蛋白的表达水平有效降低更明显；与正常组比较,激光共聚焦显微镜观察到基因敲减组细胞的线粒体部分发生断裂,碎片较多,差异有统计学意义(P＜0.05)。结论:成功构建了人源的PINK1基因敲减的质粒,并将敲减的质粒成功转染至SHSY-5Y细胞中,细胞内PINK1基因的mRNA和蛋白的表达水平降低,且细胞线粒体的形态发生了改变。这为我们后期探索PINK1基因对帕金森病线粒体功能的影响,为抗帕金森病药物的筛选及机制研究提供有力的模型基础。 |
| 英文摘要: |
| Objective: To construct and screen the human targeting specific PINK1-shRNA knockdown plasmid, transfect SH-SY5Y cells and verify the knockdown efficiency of PINK1 gene after transfection, and observe the effect on the morphology of cell mitochondria, so as to provide a cell model and experimental basis for further exploring the possible role of PINK1 gene in the pathogenesis of Parkinson""s disease. Methods: two pairs of human PINK1-shRNA sequences (numbered PINK1-shRNA-39 and PINK1-shRNA-42) were constructed, and the two pairs of interference sequences were connected to the vector to form a recombinant vector. After sequencing, the empty vector and two pairs of knockdown plasmids were transfected into SH-SY5Y cells, respectively, and the cell model of gene knockdown was obtained. The cell survival rate was detected by CCK-8 method, and the knockout efficiency of PINK1 gene was determined by fluorescence quantitative PCR. Western blotting was used to verify whether the expression level of PINK1 in cells changed, and laser confocal microscope was used to observe whether the morphology of mitochondria changed. Results: the sequencing results of the plasmid we extracted showed that the plasmid vector was successfully constructed. After transfection, the cell survival rate was decreased by CCK8 assay. Compared with the normal group, the cell survival rate of PINK1-shRNA-39 and PINK1-shRNA-42 knockdown plasmids groups decreased by 13.7 %（P＜0.05）and 14.1 %（P＜0.05）, respectively. The results of fluorescence quantitative PCR showed that compared with the normal group, the expression of PINK1 gene in the cells transfected with PINK1-shRNA-39 and PINK1-shRNA-42 knockdown plasmids decreased by 24.1 % (P < 0.01) and 36.7 % (P < 0.01), respectively. The results of Western blotting showed that compared with the normal group, the expression level of PINK1 protein in the cells transfected with PINK1-shRNA39 and PINK1-shRNA-42 knockdown plasmid decreased significantly after the two pairs of plasmids were transfected respectively, and the expression level of PINK1 protein in the cells transfected with PINK1-shRNA-42 knockdown plasmid was more significantly lower than that in the normal group. Compared with the normal group, the mitochondrial part of the cells in the gene knockout group was broken and more fragments were observed by laser confocal microscope, and the difference was statistically significant (P < 0.05). Conclusion: The human PINK1 gene knockdown plasmid was constructed successfully, and the knodown plasmid was successfully transfected into SHSY-5Y cells, and the mRNA and protein expression levels of PINK1 gene in SHSY-5Y cells were decreased, and the morphology of mitochondria changed. This provides a strong model basis for us to explore the effect of PINK1 gene on the mitochondrial function of Parkinson""s disease and to provide a strong model basis for the screening and mechanism study of anti-Parkinson""s disease drugs.
Keywords: PINK1 gene shRNA plasmid, SH-SY5Y cells, mitochondrial dysfunction, Parkinson""s disease |
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