p33^(ING1b)真核表达载体的构建及其对肝癌细胞生长的抑制作用

Construction of pCDNA3/p33^(ING1b) plasmid and its inhibitory role in the proliferation of hepatoma cell line

目的 构建pCDNA3/ p33ING1b真核表达质粒并探讨其对肝癌细胞SMMU772 1生长的抑制作用。方法 将 p33ING1bcDNA正反义亚克隆至pCDNA3真核表达载体上 ,并经磷酸钙共沉淀法分别转染至SMMU772 1细胞中。转染后 48h,用 0 .8g/LG41 8筛选出阳性克隆 ,检测转染后细胞的生长曲线和软琼脂克隆形成率的变化及应用流式细胞仪分析细胞凋亡率的改变。结果 重组 pCDNA3/ p33ING1b正反义表达质粒构建成功。经正义 p33ING1b质粒转染的SMMU772 1细胞生长速度减慢 ,快速增长期比对照组推迟 2d ,软琼脂克隆形成率为 (69.0± 1 2 .0 )‰ ,较转染空载体(90 .0± 1 0 .5)‰及反义表达质粒 (1 4 9.0± 1 5 .0 )‰的SMMU772 1细胞减少 ,细胞凋亡率 (2 2 .53 % )亦比空载体组 (8.95 % )及反义组 (7.75 % )上升。结论 重组pCDNA3/ p33ING1b质粒能在SM MU772 1细胞内表达 ,且能抑制SMMU772

Objective To construct pCDNA3/p33 ING1b eukaryotic expression plasmid and to investigate its inhibitory role in the proliferation of hepatoma cell line SMMU7721.Methods Sense and antisense p33 ING1b cDNA was subcloned into pCDNA3 eukaryotic expression vector and the recombinant plasmids were transfected into SMMU7721 cell line by calcium phosphate co precipitation method.After transfection,the cell growth curve and soft agar cloning efficiency were analyzed.Flow cytometry were used to investigate the cell apoptotic rate of transfected SMMU7721 cells.Results Sense and antisense p33 ING1b eukaryotic expression plasmid were obtained.The cell growth rate of the SMMU7721 cells transfected with sense pCDNA3/p33 ING1b was decreased.Cloning efficiency in soft agar of the cells (69.0±12.0)‰ was lower than that of cells transfected with pCDNA3 plain vector (90.0±10.5)‰ or the antisense vector(149.0±15.0)‰(P<0.01).FCM showed that the apoptotic rate of SMMU7721 cells transfected with sense p33 ING1b (22.53%)were increased.Conclusion Recombinant pCDNA3/p33 ING1b plasmid could be expressed in SMMU7721 cells and it could inhibit the proliferation of SMMU7721 cells.