慢病毒介导LIGHT基因在结直肠癌细胞HCT116中的表达  

Construction and identification of recombinant human LIGHT lentiviral vector and its expression inhuman colorectal carcinoma cells

作　　者：王海波[1] 隋爱华[2] 刘世海[2] 骆铮[1] 李传智[1] 刘相萍[2] 

机构地区：[1]青岛大学医学院附属医院普外科,青岛266003 [2]青岛大学医学院附属医院中心实验室,青岛266003

出　　处：《中华实验外科杂志》2013年第3期441-443,共3页Chinese Journal of Experimental Surgery

基　　金：山东省自然科学基金资助项目（Y2008C48）

摘　　要：目的观察慢病毒介导的LIGHT基因对结直肠癌细胞HCTll6的作用。方法将LIGHT基因克隆至慢病毒表达载体中，在293T细胞中进行病毒的包装。慢病毒以不同的感染复数（MOI）感染HCT116细胞，通过绿色荧光蛋白的表达和细胞活性筛选最适MOI。重组慢病毒感染HCT116细胞，实时荧光定量聚合酶链反应（Real—timePCR）和酶联免疫吸附试验（ELISA）法检测细胞中LIGHT基因和蛋白的表达。噻唑蓝（MTT）实验检测细胞的生长活性。结果经酶切和测序鉴定证实重组慢病毒载体构建成功。重组慢病毒的滴度为1．96x10^8TU／ml。慢病毒MOI为2感染HCT116，转染效率可达92％。慢病毒感染24、48、72、120h后，细胞中LIGHTmRNA的表达量分别是空白对照组的19．03、68．59、94．35和q1．71倍，明显高于空白对照组和慢病毒对照组（P〈0．05）；并且重组慢病毒组细胞中LIGHT蛋白含量从12h的0增加到72h的11.36μg／L，其相对生长增殖率也显著低于慢病毒对照组和空白对照组（P〈0．05）。结论成功构建LIGHT基因过表达慢病毒载体；重组慢病毒可有效转染结直肠癌细胞HCT116，高效表达LIGHT蛋白，并对HCT116细胞有明显抑制作用。Objective To construct and identify recombinant human LIGHT lentiviral vector pLenti-LIGHT and observe its expression in human colorectal carcinoma ceils HCT116. Methods The full length of human LIGHT gene was cloned to lentiviral expression vector by recombinant DNA technology. The positive clones were confirmed by enzyme digestion and DNA sequencing. The recombined lentiviriral particles were produced in 293T cells. The titer of recombinant viruses was tested, and the recombinant vi- ruses were used to transfect into HCT116 cells in different muhiple of infection （MOI） by detecting the re- porter gene expression. The LIGHT expression was determined by using real-time quantitative polymerase chain reaction （PCR） and enzyme linked immunosorbent assay （ELISA）. Cellular proliferation inhibitory activity was determined by using methyl thiazolyl tetrazolium （MTI＇） assay. Results Enzyme digestion and DNA sequencing showed that the LIGHT gene was inserted into the lentiviral vector, correctly. With th~.ti- ter of 1.96x10^8 TU/ml, the optimal MOI of the recombined lentivirus for HCT116 was 2 and the transfec- tion efficiency was up to 92% at the same time. As compared Mth the control group, the mRNA ratio of LIGHT to GAPDH in LIGHT-transfected group was increased 19.03, 68.59, 94. 35 and 11.71 folds at 24, 48, 72, 120 h respectively after transfection （P 〈 0. 05 ）, respectively. There was no significant difference between lentivirus control group and control group （P 〉 0. 05 ）. In LIGHT-transfected group, the LIGHT protein expression was significantly increased from 0 to I 1.36 tLg/L at 12 h and 72 h. MTT result revealed the cellular proliferation activity in LIGHT-transfected group was also inhibited. Conclusion The recombi- nant LIGHT lentivirus expressing vector was successfully constructed. The exogenous gene LiGHT can be expressed efficiently in HCTll6 cells and may inhibit the growth of HCT116 cells.

关 键 词：结直肠癌 慢病毒 基因转染 

分 类 号：R735[医药卫生—肿瘤]