重组pEGFP-C1-CXCL1真核表达载体构建及其对内质网应激下人肝癌细胞HepG2的增殖作用  

Construction of pEGFP-C1-CXCL1 plasmid and detection of its expression in eukaryotic cells

作　　者：毛瑞涛 陈伟[2] 李自慧[3] 邹岭[3] 叶甲舟[3] 白涛[3] 陈洁[3] 陈健康 王翠 刘宁 杨晓莉[4] 魏从文[5] 钟辉[5] 吴飞翔[3] MAO Rui-tao;CHEN Wei;LI Zi-hui;ZOU Ling;YE Jia-zhou;BAI Tao;CHEN Jie;WANG Cui;LIU Ning;YANG Xiao-li;WEI Cong-wen;ZHONG Hui;WU Fei-xiang(China Japan Union Hospital, Jilin University, Changchun 130000, China;Affiliated Hospital of Xiangnan Universty, Chenzhou, Hunan 423000, China;Department of Hepatobiliary Surgery, Affiliated Tumor Hospital, Guangxi Medical University, Nanning 530021, China;Department of Clinical Laboratory, General Hospital of Chinese People＇s Armed Police Force, Beijing 100039, China;Institute of Biotechnology, Academy of Military Medical Sciences, Beijing 100850, China)

机构地区：[1]吉林大学中日联合医院,长春130000 [2]湘南学院附属医院,湖南郴州423000 [3]广西医科大学附属肿瘤医院肝胆外科,南宁530021 [4]中国人民武装警察部队总医院检验科,北京100039 [5]军事医学科学院生物工程研究所,北京100850

出　　处：《军事医学》2017年第10期792-795,共4页Military Medical Sciences

基　　金：广西卫生和计划生育委员会重点课题资助项目(S201513);区域性高发肿瘤早期防治重点实验室子课题资助项目(GKZ201604);广西科学技术厅重点研发课题资助项目(桂科AB16380242)

摘　　要：目的构建pEGFP-C1-CXCL1真核表达载体,研究过表达CXCL1对内质网应激(ERS)下肝癌细胞增殖作用。方法以Hep G2细胞的c DNA文库为模板,通过PCR扩增CXCL1编码序列,并将其插入pEGFP-C1空载体,构建pEGFP-C1-CXCL1真核表达载体,随后将其瞬时转染到293T细胞中,通过荧光显微镜和蛋白质免疫印迹法检测CXCL1在真核细胞中的表达情况,将pEGFP-C1-CXCL1转染至Hep G2细胞中,CCK8检测CXCL1对TM诱导下的ERS肿瘤增殖的抑制作用。结果菌液PCR和测序结果均证实得到pEGFP-C1-CXCL1真核表达载体,其转染293T细胞后,在荧光显微镜下可观察到绿色荧光,免疫印迹表明CXCL1在293T细胞中表达,CCK8实验表明,ERS条件下肿瘤的增殖受到抑制,而过表达的CXCL1可降低ERS下的Hep G2细胞的增殖抑制。结论成功构建了真核表达质粒pEGFP-C1-CXCL1,并在人胚肾293T细胞中瞬时表达成功,过表达的CXCL1可有效降低由ERS造成的肝癌细胞的增殖抑制。Objective To construct the pEGFP-C1-CXCL1 eukaryotic expression vector and to investigate the effect of CXCL1 on the proliferation of Hep G2 cells under endoplasmic reticulum stress（ ERS）. Methods Fragments of CXCL1 were obtained from the c DNA library of Hep G2 cells before CXCL1 was cloned into a pEGFP-C1 vector for a recombinant plasmid pEGFP-C1-CXCL1 which was screened and identified by PCR and sequence alignment. Then,the recombinant plasmid of pEGFP-C1-CXCL1 was transfected into human 293T cell line and the expression of CXCL1 was detected by fluorescence microscopy and Western blotting. pEGFP-C1-CXCL1 was furhter transfected into Hep G2 cells,and CCK8 was used to detect the inhibitory effect of CXCL1 on tumor proliferation induced by TM in hepatocellular carcinoma. Results pEGFPC1-CXCL1 was vertified by sequencing analysis. Fluorescence microscopy showed that pEGFP-C1-CXCL1 was transfected into 293T. CXCL1 expression was detected by Western blotting. CCK8 showed that TM inhibited tumor proliferation,while overexpression of CXCL1 decreased the inhabitory rate on cell proliferation of Hep G2 cells under ER stress compared to pEGFP-C1 group and the control group. Conclusion A recombinant pEGFP-C1-CXCL1 plasmid is successfully constructed that can be expressed stably in human 293T cells. Overexpression of CXCL1 can effectively reduce the inhabitory rate of HCC cells induced by the ER stress.

关 键 词：趋化因子类 CXCL1 重组质粒 细胞增殖 癌 肝细胞 

分 类 号：R735.7[医药卫生—肿瘤]