Seipin敲低大鼠PC12细胞株的构建和对细胞凋亡的影响  

Construction of Rat PC12-Seipin Knockdown Cell Line and its Effect on Apoptosis

作　　者：吕菊 胡玉梅 任真奎 谢鹏 张春林 焦玲[5] 禹文峰[1,2] LV Jv;HU Yumei;REN Zhenkui;XIE Peng;ZHANG Chunlin;JIAO Ling;YU Wenfeng(Key Laboratory of Molecular Biology of Guizhou Medical University,Guiyang 550004,Guizhou,China;Education Ministry Key Laboratory of Endemic and Minority Diseases,Guizhou Medical University,Guiyang 550004,Guizhou,China;Department of Laboratory,People's Hospital of Southwest Guizhou Autonomous Prefecture,Xingyi 562400,Guizhou,China;Basic Medical College,Guizhou Medical University,Guiyang 550025,Guizhou,China;Department of Neurology,the Affiliated Hospital of Guizhou Medical University,Guiyang 550004,Guizhou,China)

机构地区：[1]贵州医科大学分子生物学重点实验室,贵州贵阳550004 [2]贵州医科大学地方病与少数民族疾病教育部重点实验室,贵州贵阳550004 [3]黔西南州人民医院检验科,贵州兴义562400 [4]贵州医科大学基础医学院,贵州贵阳550025 [5]贵州医科大学附院神经内科,贵州贵阳550004

出　　处：《贵州医科大学学报》2020年第3期249-254,共6页Journal of Guizhou Medical University

基　　金：国家自然科学基金(81360199);教育部科学技术研究项目(2013032A);贵州省国际科技合作计划项目[黔科合外G字(2013)7026];贵州省创新计划项目[黔教合协同创新中心(2014)06];贵州省教育厅项目(2015年贵州省普通高等学校地方病和少数民族疾病防控创新团队);贵州省科技厅计划课题[黔科合重大专项字(2014)6008];贵州省教育厅项目[黔教合外G字(2013)63]。

摘　　要：目的:构建大鼠肾上腺嗜铬细胞瘤(PC12)细胞的先天性脂肪代谢障碍2型(BSCL2/Seipin)敲低细胞株,探讨Seipin敲低后对细胞凋亡的影响。方法:将PC12细胞分为空白感染组和助感染组(加助感染试剂Hitans G),采用不同慢病毒感染复数(MOI)感染细胞进行病毒预感染试验筛选助感染试剂和合适的MOI;依据上述感染条件,用阴性对照病毒和Seipin-shRNA目的病毒感染PC12细胞作为阴性对照组和Seipin敲低组,同时设置正常组(正常PC12细胞),采用蛋白质免疫印迹法检测Seipin蛋白的表达;将阴性对照组与Seipin敲低组细胞用脱氧核苷酸末端转移酶介导的d UTP缺口末端标记(TUNEL)细胞凋亡染色法和Hoechst 33258染色法检测细胞凋亡情况。结果:慢病毒MOI=100时感染效果最强,助感染试剂能增加感染效率;与正常组相比,Seipin敲低组Seipin蛋白表达水平降低(P<0.05);与阴性对照组相比,TUNEL染色结果显示Seipin敲低组发红色荧光的凋亡细胞明显增加,Hoechst染色结果显示Seipin敲低组细胞的细胞核致密浓染或碎块状致密浓染程度明显增多。结论:成功构建大鼠PC12 Seipin敲低细胞,下调Seipin蛋白表达会增加细胞凋亡。Objective:To construct rat Berardinelli-Seip congenital lipodystrophy2(PC12-BSCL2/Seipin)knockdown cell line and investigate the effect of Seipin knockdown on apoptosis.Methods:Pheochromocytoma cells 12(PC12 cells)were divided into the blank infection group and HitansG group(add HitansG agent),and received infection with different lentivirus MOI for virus pre-infection test to screen HitansG ragent and appropriate MOI.According to the infection conditions,PC12 cells received infection with negative control virus and Seipin-shRNA virus,and the control group(normal PC12 cells)was set at the same time.The level of Seipin protein was detected by Western blot.Cells of the negative control group and Seipin knockdown group was stained by TdT-mediated dUTP Nick-End Labeling(TUNEL)and Hoechst 33258 to detect apoptosis.Results:The transfection efficiency of lentivirus was the best in MOI=100,and the HitansG increased the transfection efficiency.Compared with the controls,the level of Seipin protein in the Seipin knockdown group was significantly reduced(P<0.05).Compared with the negative controls,the apoptosis results of TUNEL staining showed a significant increase in red fluorescent apoptotic cells in the Seipin knockdown group,and the Hoechst 33258 staining showed condensed bright nuclei apoptotic cells in the Seipin knockdown group.Conclusion:PC12 Seipin knockdown cells are successfully constructed,and down-regulation of Seipin protein can increase apoptosis.

关 键 词：细胞凋亡 帕金森病 PC12细胞 神经变性疾病 Seipin蛋白 基因干扰 

分 类 号：R742.5[医药卫生—神经病学与精神病学] R-332[医药卫生—临床医学]