Seipin变化对6-OHDA诱导的PC12帕金森细胞模型自噬及凋亡的影响  

Effect of Seipin expression on autophagy and apoptosis of 6-OHDA-induced Parkinson disease cellular model

作　　者：胡玉梅 郭冬芬 任真奎 刘颖[1,2] 吴昌学[1,2] 禹文峰[1,2,3] HU Yumei;GUO Dongfen;REN Zhenkui;LIU Ying;WU Changxue;YU Wenfeng(Key Laboratory of Molecular Biology,Guizhou Medical University,Guiyang 550004,Guizhou,China;Education Ministry Key Laboratory of Endemic and Minority Diseases,Guizhou Medical University,Guiyang 550004,Guizhou,China;School of Basic Medical Science,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]贵州医科大学基础医学院,贵州贵阳550004

出　　处：《贵州医科大学学报》2021年第4期392-397,409,共7页Journal of Guizhou Medical University

基　　金：国家自然科学基金(81360199);中央引导地方科技发展专项资金[黔科中引地(2019)4008];贵州省卫生健康委科学技术基金(gzwjkj2019-1-039);黔西南州科技局基金(2019-1-10)。

摘　　要：目的探究Seipin蛋白对6-OHDA诱导的PC12帕金森细胞模型凋亡及自噬的影响。方法PC12细胞分为正常组和加药组(加入6-OHDA构建帕金森模型细胞);将前期构建成功的Seipin敲低及其敲低对照细胞株、Seipin过表达及其过表达对照细胞株分为Seipin敲低对照组(KD-control)、Seipin敲低组(KD)、Seipin敲低对照加药组(KD-control+6-OHDA)及Seipin敲低加药组(KD+6-OHDA),Seipin过表达对照组(OE-control)、Seipin过表达组(OE)、Seipin过表达对照加药组(OE-control+6-OHDA)、Seipin过表达加药组(OE+6-OHDA);采用Western blot检测Seipin、哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)、磷酸化mTOR(p-mTOR)、自噬相关蛋白Beclin1、微管相关蛋白1A/1B-轻链3(LC3-1A/B)及凋亡相关蛋白BCL2-Associated X蛋白质(Bax)、B淋巴细胞瘤-2(Bcl-2)、裂解型半胱氨酸水解酶3(Cleved caspase-3)蛋白表达水平。结果6-OHDA处理的PC12细胞中,与正常组比较,6-OHDA处理后Seipin表达降低、p-mTOR/mTOR的比值降低、Beclin1、LC3-Ⅱ/LC3-Ⅰ、Bax/Bcl-2、Cleved caspase-3的表达升高(P<0.01);在Seipin敲低后,KD相较于KD-control,p-mTOR/mTOR的比值升高、自噬相关蛋白Beclin1及LC3-Ⅱ/LC3-Ⅰ的表达均降低,凋亡相关蛋白Bax/Bcl-2和Cleved caspase-3表达均升高(P<0.05),过表达Seipin与敲低Seipin结果相反;加药处理后KD+6-OHDA相对KD-control+6-OHDA,Seipin含量降低,p-mTOR/mTOR的比值升高、自噬相关蛋白Beclin1及LC3-Ⅱ/LC3-Ⅰ的表达均降低,凋亡相关蛋白Bax/Bcl-2和Cleved caspase-3表达均升高(P<0.05);在Seipin过表达组中,过表达Seipin与敲低Seipin结果相反。结论6-OHDA诱导的细胞损伤可能是通过减少Seipin蛋白的表达实现,上调Seipin蛋白的表达可对6-OHDA诱导的细胞损伤具有抑制作用。Objective To investigate the effect of Seipin expression on autophagy and apoptosis of 6-OHDA-induced parkinson disease(PD)cellular model.Methods PC12 cells was divided into normal group and 6-OHDA group.6-OHDA treatment was used to establish PD cellular model.Based on treatment with or without 6-OHDA,previously generated PC12 cells with Seipin knockdown(KD)or KD-control,with Seipin overexpression(OE)or OE control were divided into 8 groups:Seipin KD,KD-control,Seipin KD+6-OHDA,KD-control+6-OHDA,OE,OE control,OE+6-OHDA,and OE control+6-OHDA.Western blot was used to detect the protein expression levels of Seipin,mammalian target of rapamycin(mTOR),phosphorylated mTOR(p-mTOR),and autophagy-related protein Beclin1 and microtubule-associated protein 1A/1B-light chain 3(LC3-1A/B),apoptosis-associated protein Bax,B lymphoma-2(Bcl-2),and Cleved caspase-3.Results When compared to normal group,6-OHDA treatment reduced Seipin expression,p-mTOR/mTOR ratio,but increased the expression levels of Beclin1,LC3-Ⅱ/LC3-Ⅰ,Bax/Bcl-2,and Cleved caspase-3 in PC12 cells(P<0.01).When compared to KD control group,silencing Seipin upregulated p-mTOR/mTOR ratio,decreased autophagy-related Beclin1 and LC3-Ⅱ/LC3-Ⅰand increased apoptosis-related Bax/Bcl-2,and Cleved caspase-3.The effect of overexpressing Seipin on these protein expression levels was contrary to silencing Seipin.When compared to KD-control,6-OHDA treatment significantly reduced Seipin expression,upregulated p-mTOR/mTOR ratio,decreased autophagy-related Beclin1,and LC3-Ⅱ/LC3-Ⅰand increased apoptosis-related Bax/Bcl-2,and Cleved caspase-3(P<0.05).The effect of overexpressing Seipin on these protein expression levels was contrary to silencing Seipin after 6-OHDA treatment.Conclusion 6-OHDA-induced cell damage may be achieved by reducing Seipin protein expression.Overexpressing Seipin may have an inhibitory effect on 6-OHDA-induced cell damage.

关 键 词：帕金森 6-羟基多巴胺氢溴酸盐 Seipin 自噬 凋亡 

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]