CAJ | 학술논문

目的观察ADP核糖化因子鸟苷酸激酶1(ASAP1)对胃癌细胞株SGC7901增殖、迁移能力的影响.方法构建靶向过表达ASAP1基因的慢病毒载体LV-ASAP1-GFP,感染胃癌SGC7901细胞株,经过筛选,建立稳定过表达ASAP1基因的胃癌SGC7901细胞株.通过实时定量聚合酶链反应(Real-time PCR)、Western blot分别检测ASAP1基因mRNA与蛋白表达量,噻唑蓝(MTT)比色法、Transwell体外细胞迁移实验分别研究稳定过表达ASAP1基因对细胞增殖和迁移的影响.结果成功包装靶向过表达ASAP1基因的慢病毒载体LV-ASAP1-GFP稳定过表达ASAP1基因的人胃癌SGC7901细胞.与空载对照LV-GFP组和阴性SGC7901细胞组比较,Real-time PCR与Western blot证实,慢病毒LV-ASAP1-GFP组mRNA相对表达量为对照组的255.4％,蛋白相对表达量为对照组的290.2％;MTT比色法显示LV-ASAP1-GFP组细胞增殖能力增强,在1、2、3d后重复检测,分别达到了同时间空白对照组的130.1％、136.0％和149.1％ (P ＜0.05);细胞迁移实验显示LV-ASAP1-GFP组细胞迁移能力显著增强,穿膜细胞数为对照组的230.3％ (P＜0.01).结论慢病毒介导稳定过表达ASAP1基因能使胃癌SGC7901细胞株的增殖能力及迁移能力增强.
목적 관찰ADP핵당화인자조감산격매1(ASAP1)대위암세포주SGC7901증식、천이능력적영향.방법 구건파향과표체ASAP1기인적만병독재체LV-ASAP1-GFP,감염위암SGC7901세포주,경과사선,건립은정과표체ASAP1기인적위암SGC7901세포주.통과실시정량취합매련반응(Real-time PCR)、Western blot분별검측ASAP1기인mRNA여단백표체량,새서람(MTT)비색법、Transwell체외세포천이실험분별연구은정과표체ASAP1기인대세포증식화천이적영향.결과 성공포장파향과표체ASAP1기인적만병독재체LV-ASAP1-GFP은정과표체ASAP1기인적인위암SGC7901세포.여공재대조LV-GFP조화음성SGC7901세포조비교,Real-time PCR여Western blot증실,만병독LV-ASAP1-GFP조mRNA상대표체량위대조조적255.4％,단백상대표체량위대조조적290.2％;MTT비색법현시LV-ASAP1-GFP조세포증식능력증강,재1、2、3d후중복검측,분별체도료동시간공백대조조적130.1％、136.0％화149.1％ (P ＜0.05);세포천이실험현시LV-ASAP1-GFP조세포천이능력현저증강,천막세포수위대조조적230.3％ (P＜0.01).결론 만병독개도은정과표체ASAP1기인능사위암SGC7901세포주적증식능력급천이능력증강.
Objective To construct a stable over-expression lentivirus-mediated vector and transfect it into human gastric cancer cell line SGC7901 for investigating its effects on proliferation and migration of GC7901 cells.Methods ADP ribosylation factor guanylate kinase 1 (ASAP1) gene coding region was cloned into lentivirus vector.Lentivirus particles were infected into the human gastric carcinoma cell line SGC7901 to upregulate the expression of ASAP1 gene.The up-regulated efficiency of targeting ASAP1 gene at mRNA level was detected by real-time quantitative polymerase chain reaction (Real-time PCR),the effect on proliferation of mesenchmal stem cells was assayed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay,and the migration ability was detected by Transwell motility assay.Results The lentiviral vector targeting ASAP1 gene was constructed successfully,and a stable human gastric cancer cell line SGC7901 line that up-regulated ASAP1 was established.Real-time quantitative PCR and Western blotting results showed that the expression of ASAP1 gene was efficiently up-regulated by infecting LV-ASAP1-GFP (P ＜ 0.01).255.4％ of control group on mRNA expression and 290.2％ of control group on protein expression.The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and growth curve showed that the over-expression of the ASAP1 gene successfully increased the proliferative capability of SGC7901 cells,with optical density values reaching 130.1％,136.0％ and 149.1％ of control group at 1,2,3 d respectively.The Transwell assay also showed similar increasing results on the migration ability (P ＜ 0.01).Conclusion The lenvivirus-mediated over-expression of the ASAP1 gene increases the proliferation and migration abilities of human gastric cancer cell line GC7901.