稳定表达Cytoglobin的肝细胞株LO-2的建立及生长增殖的变化  

Establishment of hepatocyte LO-2 cell line stably expressing Cytoglobin and its changes in proliferation

作　　者：王哲彦 陈柏洪 续艳梅 刘诗俐 张梓荣 董文其 WANG Zheyan;CHEN Bohong;XU Yanmei;LIU Shili;ZHANG Zirong;DONG Wenqi(Department of laboratory medicine and Biotechnology,Southern Medical University,Guangdong 510080,China;Department of Life Sciences,Sun Yat-sen University,Guangdong 510275,China)

机构地区：[1]南方医科大学检验与生物技术学院,广东广州510080 [2]中山大学生命科学学院,广东广州510275

出　　处：《暨南大学学报（自然科学与医学版）》2020年第1期70-78,共9页Journal of Jinan University(Natural Science & Medicine Edition)

基　　金：广东省省级科技计划项目(2013A022100027)。

摘　　要：目的:建立过表达细胞珠蛋白Cytoglobin(CYGB)的人肝细胞LO-2稳定株,初步探究CYGB对LO-2细胞生长增殖的影响.方法:采用PCR法扩增CYGB编码基因,并通过GATEWAY克隆技术构建慢病毒重组表达载体Plenti-N-GFP-CYGB;酶切、测序鉴定后,与包装质粒三质粒共转人肾上皮细胞(HEK293T),收集、浓缩含病毒上清,获得病毒颗粒;感染LO-2细胞并采用流式细胞分选技术筛选稳定细胞株,Western blot检测表达情况;使用CCK-8试剂盒检测过表达CYGB对LO-2细胞增殖的影响,通过流式细胞技术检测过表达CYGB对LO-2细胞凋亡的影响.结果:慢病毒重组表达载体Plenti-N-GFP-CYGB双酶切鉴定以及基因序列比对鉴定均正确.三质粒共转HEK293T细胞后,荧光显微镜下观察大部分细胞出现绿色荧光;收集病毒颗粒并感染LO-2细胞后,经流式细胞分选技术筛选获得稳定表达CYGB的细胞,Western blot鉴定显示,LO-2稳定株组的CYGB表达条带明显,阴性对照组未出现条带.CCK-8法绘制的细胞生长曲线显示,稳定表达细胞组的细胞生长速率低于阴性对照组,有统计学差异(P<0.01).流式细胞技术检测各组LO-2细胞凋亡情况结果显示,稳定表达细胞组的细胞凋亡百分比高于阴性对照组(P<0.05).结论:成功构建慢病毒重组表达载体Plenti-N-GFP-CYGB;建立稳定表达CYGB的人肝细胞株LO-2-GFP-CYGB以及阴性对照组细胞株LO-2-GFP;稳定表达CYGB的肝细胞生长速率较阴性对照组肝细胞降低,稳定表达CYGB的肝细胞凋亡百分比高于阴性对照组肝细胞;为CYGB与其蛋白的相互关系研究提供了细胞模型.Objective:To establish a stable human hepatocyte LO-2 strain overexpressing Cytoglobin(CYGB)and to investigate the effect of CYGB on the growth and proliferation of LO-2 cells.Methods:The CYGB gene was amplified by PCR and the lentiviral recombinant expression vector Plenti-N-GFP-CYGB was constructed by Gateway cloning.After digestion and sequencing,the plasmid was co-transfected with HEK293T cells.The virus-containing supernatant was collected and concentrated,virus particles were obtained,LO-2 cells were infected,and stable cell lines were screened by flow cytometry.Western Blot was used to detect the expression,and the effect of overexpression of CYGB on proliferation of LO-2 cells was detected using CCK8 kit.The effect of overexpression of CYGB on apoptosis of LO-2 cells was detected by flow cytometry.Results:The results of the restriction enzyme digestion of the lentiviral recombinant expression vector Plenti-N-GFP-CYGB and the alignment of the gene sequences were correct.After the three plasmids were co-transfected into HEK293T cells,most of the cells showed green fluorescence under fluorescence microscope.After collecting the virus particles and infecting LO-2 cells,the cells stably expressing CYGB were screened by flow cytometry,and Western Blot showed that the CYGB expression bands of the LO-2 stable strain group were obvious,and no band appeared in the negative control group.The cell growth curve showed that the cell growth rate of the stable expression group was lower than that of the control group,Statistically different(P<0.01).Flow cytometry analysis showed that the percentage of apoptosis in the stable expression group was higher than that in the control group(P<0.05).Conclusion:The lentiviral recombinant expression vector Plenti-N-GFP-CYGB was successfully constructed.The human liver cell line LO-2-CYGB stably expressing CYGB was established.The growth rate of hepatocytes stably expressing CYGB was lower than that of the normal cell group.The percentage of hepatocyte apoptosis stably expressing CY

关 键 词：CYGB LO-2 慢病毒重组表达载体 稳定表达 细胞凋亡 

分 类 号：Q291[生物学—细胞生物学]