CAJ | 학술논문

目的观察慢病毒介导的LIGHT基因对结直肠癌细胞HCT116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-time PCR)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1.96×108 TU/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、120 h后,细胞中LIGHT mRNA的表达量分别是空白对照组的19.03、68.59、94.35和-11.71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12 h的0增加到72 h的11.36μ g/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.
목적 관찰만병독개도적LIGHT기인대결직장암세포HCT116적작용.방법 장LIGHT기인극륭지만병독표체재체중,재293T세포중진행병독적포장.만병독이불동적감염복수(MOI)감염HCT116세포,통과록색형광단백적표체화세포활성사선최괄MOI.중조만병독감염HCT116세포,실시형광정량취합매련반응(Real-time PCR)화매련면역흡부시험(ELISA)법검측세포중LIGHT기인화단백적표체.새서람(MTT)실험검측세포적생장활성.결과 경매절화측서감정증실중조만병독재체구건성공.중조만병독적적도위1.96×108 TU/ml.만병독MOI위2감염HCT116,전염효솔가체92％.만병독감염24、48、72、120 h후,세포중LIGHT mRNA적표체량분별시공백대조조적19.03、68.59、94.35화-11.71배,명현고우공백대조조화만병독대조조(P＜0.05)；병차중조만병독조세포중LIGHT단백함량종12 h적0증가도72 h적11.36μ g/L,기상대생장증식솔야현저저우만병독대조조화공백대조조(P＜0.05).결론 성공구건LIGHT기인과표체만병독재체；중조만병독가유효전염결직장암세포HCT116,고효표체LIGHT단백,병대HCT116세포유명현억제작용.
Objective To construct and identify recombinant human LIGHT lentiviral vector pLenti-LIGHT and observe its expression in human colorectal carcinoma cells 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 viruses were used to transfect into HCT116 cells in different multiple of infection (MOI) by detecting the reporter 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 (MTT) assay.Results Enzyme digestion and DNA sequencing showed that the LIGHT gene was inserted into the lentiviral vector,correctly.With thetiter of 1.96 × 108 TU/ml,the optimal MOI of the recombined lentivirus for HCT116 was 2 and the transfection efficiency was up to 92％ at the same time.As compared with 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 11.36 μg/L at 12 h and 72 h.MTT result revealed the cellular proliferation activity in LIGHT-transfected group was also inhibited.Conclusion The recombinant LIGHT lentivirus expressing vector was successfully constructed.The exogenous gene LIGHT can be expressed efficiently in HCT116 cells and may inhibit the growth of HCT116 cells.