稳定沉默Beclin1的人成神经瘤SH-SY5Y细胞系的构建  

Establishment of Human Neuroblastoma SH-SY5Y Cell Line Stably Silencing Beclin1

作　　者：魏传杰[1] 肖双[1] 江岚[1] 谭琰[1] 黄波[1] 龙鼎新[1] 

机构地区：[1]南华大学公共卫生学院,衡阳421001

出　　处：《生物医学工程学杂志》2014年第5期1085-1089,共5页Journal of Biomedical Engineering

基　　金：国家自然科学基金资助项目(81172712);湖南省自然科学基金资助项目(11JJ6078);湖南省教育厅优秀青年基金资助项目(09B087);南华大学博士启动基金资助项目(2010XQD19)

摘　　要：为了构建pGenesil-1-Beclin1真核表达载体,并建立Beclin1稳定沉默的人成神经瘤SH-SY5Y细胞系,将合成的shRNA片段连接pGenesil-1质粒,构建成重组质粒pGenesil-1-Beclin1,转化其至感受态大肠杆菌JM109,挑取阳性克隆进行双酶切和DNA测序鉴定。常规培养SH-SY5Y细胞,将重组质粒pGenesil-1-Beclin1和空白对照质粒pGenesil-1转染SH-SY5Y细胞,G418抗性筛选,在荧光显微镜下挑取单细胞克隆并扩大培养至稳定细胞株,经RT-PCR和Western blot检测Beclin1基因的表达。经酶切和DNA测序鉴定重组真核表达载体构建正确,成功筛选出两株细胞系。和对照组转染pGenesil-1的细胞系相比,转染pGenesil-1-Beclin1细胞系的Beclin1mRNA表达下降71.28%±1.45%(P<0.05),Beclin1蛋白表达下降75.50%±2.63%(P<0.05)。提示已经成功构建pGenesil-1-Beclin1真核表达载体并建立了Beclin1基因稳定沉默的SH-SY5Y细胞系,为研究Beclin1功能提供了细胞模型。The pGenesil-1-Beclin1 eukaryotic expression vectors were constructed to establish an SH-SY5 Ycell line stably expressing shRNA-Beclin1.The shRNA was connected to pGenesil-1to construct the recombinant plasmid pGenesil-1-Beclin1,which was transformed into JM109 E.coli.Positive clones were identified by digestion with restriction endonuclease and DNA sequencing.SH-SY5 Ycells were cultured by the conventional method.The pGenesil-1-Beclin1 and pGenesil-1plasmids were transfected into SH-SY5 Ycells,and the cells were screened by G418 until the stable G418-resistant monoclonal cells were acquired.Beclin1 mRNA and Beclin1 protein were detected by RTPCR and Western blot analysis respectively.The results of restriction endonuclease analysis and DNA sequencing confirmed the correct construction of the eukaryotic expression vector pGenesil-1-Beclin1.Two SH-SY5 Ytransfected cell lines were successfully selected.Compared with the control group,RT-PCR and Western blot showed that the expression of Beclin1 mRNA and protein were down regulated 71.28%±1.45%（P〈0.05）and 75.50%±2.63%（P〈0.05）,respectively.The results indicated that the eukaryotic expression vector pGenesil-1-Beclin1 was successfully constructed and the SH-SY5 Ycell lines with inhibited Beclin1 expression were established.It provides a useful cell model for studying the biological function of Beclin1.

关 键 词：JM109 SH-SY5Y pGenesil-1-Beclin1 G418筛选 细胞克隆 

分 类 号：Q78[生物学—分子生物学]