摘要
目的:采用基因沉默和慢病毒转染技术,沉默人肝癌细胞HepG2中特异蛋白3(specificity protein 3,Sp3)的表达,观察沉默Sp3基因后的肝细胞的增殖能力变化.方法:采用Sp3-siRNA慢病毒转染人肝癌细胞HepG2,通过免疫印迹法和PCR技术检测Sp3的表达以验证转染效果,通过MTT检测细胞生长曲线和流式细胞技术测定细胞周期.结果:Western blot实验表明,实验组的Sp3表达量明显少于空白组和阴性对照组(0.37±0.08 vs 0.83±0.17,0.66±0.13,F=8.442,均P<0.05).RT-PCR也得到相同的结果(0.47±0.05 vs 0.74±0.08,0.70±0.16,F=7.322,均P<0.05).MTT实验结果显示,与空白对照和阴性对照组相比,实验组细胞在48、72和96h时的增殖明显受到抑制(0.28±0.18 vs 0.34±0.19,0.35±0.07,F=3.888;0.57±0.11 vs 0.84±0.05,0.74±0.08,F=12.721;0.72±18.1 vs 0.98±0.05,0.93±0.9,F=6.342,均P<0.05).流式细胞术结果显示实验组细胞主要分布在G1期.结论:RNAi沉默Sp3基因导致人肝癌HepG2细胞体外增殖能力下降.
AIM：To investigate the effect of RNA interference（RNAi）-mediated gene silencing of specif icity protein 3（Sp3） on the proliferation of human hepatocellular carcinoma HepG2 cells.METHODS：HepG2 cells were infected with a lentivirus expressing Sp3-siRNA,and the ex-pression of Sp3 mRNA and protein was determined by semi-quantitative reverse transcription polymerase chain reaction（RT-PCR）,and Western blot and immunohistochemistry.Cell growth was evaluated by methyl thiazolyl tetrazolium（MTT） assay,and cell cycle progression was analyzed by fow cytometry.RESULTS：Compared to control cells,the expression levels of Sp3 mRNA and protein were signif icantly lower in HepG2 cells transfected with the Sp3-siRNA（mRNA：0.47 ± 0.05 vs 0.74 ± 0.08,0.70 ± 0.16,F = 7.322,all P 0.05;protein：0.37 ± 0.08 vs 0.83 ± 0.17,0.66 ± 0.13,F = 8.442,all P 0.05）.MTT assay showed that the growth of cells transfected with the Sp3-siRNA was slower at 48,72 and 96 h（0.28 ± 0.18 vs 0.34 ± 0.19,0.35 ± 0.07,F = 3.888;0.57 ± 0.11 vs 0.84 ± 0.05,0.74 ± 0.08,F=12.721;0.72±18.1vs0.98±0.05,0.93 ± 0.9,F = 6.342,all P 0.05）.Flow cytometry analysis showed that the percentage of cells in G1 phase increased in cells transfected with the Sp3-siRNA.CONCLUSION：Sp3 may play an important role in the growth of human hepatic cancer cells,and RNAi-induced Sp3 down-regulation could inhibit the growth of HepG2 cells in vitro.
出处
《世界华人消化杂志》
CAS
北大核心
2012年第27期2595-2600,共6页
World Chinese Journal of Digestology
基金
广西壮族自治区卫生厅自筹经费科研基金资助项目
No.Z2012052
广西壮族自治区"新世纪十百千人才工程"专项基金资助项目
No.2007214~~
