RRS1在MPP^+诱导SH-SY5Y细胞凋亡过程中表达的变化及意义  

CHANGE IN THE EXPRESSION OF REGULATOR OF RIBOSOME SYNTHESIS 1 AND ITS SIGNIFICANCE IN 1-METHYL-4-PHENYLPYRIDINE-INDUCED APOPTOSIS OF SH-SY5Y CELLS

作　　者：能一鸣 张峥[1] 李雪[1] 王芳玲 华亚男 侯琳[1] NENG Yiming;ZHANG Zheng;LI Xue;WANG Fangling;HUA Yanan;HOU Lin(Department of Biochemistry and Molecular Biology, School of Bascic Medicine, Qingdao University, Qingdao 266071, China)

机构地区：[1]青岛大学基础医学院生物化学与分子生物学系,山东青岛266071

出　　处：《精准医学杂志》2020年第4期323-327,330,共6页Journal of Precision Medicine

基　　金：国家自然科学基金面上项目(81472542)。

摘　　要：目的探究核糖体合成调节因子1(RRS1)在1-甲基-4-苯基吡啶离子(MPP^+)诱导的人神经母细胞瘤细胞SH-SY5Y凋亡过程中表达的变化及意义。方法以含有0、100、200、400μmol/L MPP^+的培养基培养SH-SY5Y细胞48 h,用CCK-8法检测各组细胞的增殖活力,用JC-1法检测细胞线粒体膜电位,采用Annexin V-APC/PI试剂盒检测细胞凋亡率,用Western blot检测细胞抑癌因子P53、Bcl-2相关X蛋白(Bax)、B淋巴细胞瘤-2蛋白(Bcl-2)与RRS1蛋白的表达。构建RRS1慢病毒过表达载体(Lv-RRS1)和慢病毒阴性对照载体,对细胞进行慢病毒转染后,用实时荧光定量PCR(qPCR)和Western blot检测慢病毒转染效率;对转染细胞以400μmol/L MPP^+处理48 h,用CCK-8检测细胞增殖活力,用Annexin V-APC/PI试剂盒检测细胞凋亡率,用Western blot检测细胞RRS1、P53、Bcl-2和Bax蛋白的相对表达情况。结果细胞经MPP^+处理培养,SH-SY5Y细胞增殖活力、线粒体膜电位显著降低(F=95.63、69.90,P<0.05);细胞凋亡率明显升高(F=258.49,P<0.01);P53蛋白和Bax蛋白表达明显升高(F=40.47、20.78,P<0.05);RRS1蛋白和Bcl-2蛋白表达明显降低(F=28.44、42.43,P<0.05)。慢病毒转染后,与对照组相比较,Lv-RRS1组RRS1 mRNA和蛋白表达量均明显增加(t=15.59、6.05,P<0.01);对转染的细胞以400μmol/L MPP^+处理48 h后,与对照组相比较,Lv-RRS1组细胞增殖活力显著升高(t=4.76,P<0.01);细胞凋亡率明显降低(t=5.80,P<0.01);RRS1蛋白和Bcl-2蛋白表达明显升高(t=12.41、3.14,P<0.05);P53蛋白和Bax蛋白表达显著降低(t=5.20、4.77,P<0.05)。结论RRS1在MPP^+诱导的SH-SY5Y细胞凋亡中低表达,RRS1可能通过影响凋亡相关因子P53、Bax和Bcl-2蛋白的表达参与MPP^+诱导的SH-SY5Y细胞的凋亡。Objective To investigate the change in the expression of regulator of ribosome synthesis 1(RRS1)and its significance in 1-methyl-4-phenylpyridine(MPP^+)-induced apoptosis of human neuroblastoma SH-SY5Y cells.Methods SH-SY5Y cells were cultured in the medium containing 0,100,200,and 400μmol/L MPP^+for 48 h;CCK-8 assay was used to mea-sure cell proliferation activity,JC-1 assay was used to measure the mitochondrial membrane potential of SH-SY5Y cells,Annexin V-APC/PI apoptosis kit was used to measure the apoptosis rate of cells,and Western blot was used to measure the protein expression of tumor suppressor P53,B-cell lymphoma-2(Bcl-2),Bcl-2-asociated X protein(Bax),and RRS1.The lentivirus-RRS1 overexpression vector(Lv-RRS1)and lentivirus-negative vector were constructed,and after the cells were transfected with lentivirus,quantitative real-time PCR and Western blot were used to measure the transfection efficiency of lentivirus;the transfected cells were treated with 400μmol/L MPP^+for 48 h,and then,CCK-8 assay was used to measure cell proliferation activity,Annexin V-APC/PI apoptosis kit was used to measure the apoptosis rate of cells,and Western blot was used to measure the protein expression of RRS1,P53,Bcl-2,and Bax.Results After MPP^+treatment,SH-SY5Y cells had significant reductions in cell proliferation activity and mitochondrial membrane potential(F=95.63,69.90,P<0.05),significant increases in apoptosis rate(F=258.49,P<0.01)and the protein expression of P53 and Bax(F=40.47,20.78,P<0.05),and significant reductions in the protein expression of RRS1 and Bcl-2(F=28.44,42.43,P<0.05).After lentivirus transfection,compared with the control group,the Lv-RRS1 group had significant increases in the mRNA and protein expression of RRS1(t=15.59,6.05,P<0.01);after the transfected cells were treated with 400μmol/L MPP^+for 48 h,compared with the control group,the Lv-RRS1 group had a significant increase in cell proliferation activity(t=4.76,P<0.01),a significant reduction in apoptosis rate(t=5.80,P<0.01),significant inc

关 键 词：核蛋白质类 1-甲基-4-苯基吡啶 细胞系 肿瘤 神经母细胞瘤 帕金森病 细胞凋亡 基因 p53 BCL-2相关X蛋白质 原癌基因蛋白质C-BCL-2 体外研究 

分 类 号：R742.5[医药卫生—神经病学与精神病学]