摘要
目的:观察过氧化物酶体增殖物活化受体γ(peroxisome proliferators-activated receptorγ,PPARγ)基因静默对肝癌细胞增殖与凋亡的影响。方法:构建靶向PPARγ的短发夹状RNA真核表达载体并转染HCCLM3细胞,采用RT-PCR、Western印迹法检测HCCLM3细胞中PPARγ的表达;MTT法检测细胞增殖;TUNEL法及FCM法检测细胞的凋亡;免疫细胞化学法检测PCNA、野生型p53的表达。结果:shPPARγ转染组PPARγmRNA表达下调80.5%。转染后48 h,pshPPARγ组细胞增殖抑制率为71.5%;40 h时,PCNA阳性率为(23.8±7.2)%;TUNEL法检测凋亡率为(24.2±4.7)%,FCM法检测凋亡率为(23.2±4.2)%,2种方法的检测结果一致;shPPARγ转染组细胞被阻滞于G0/G1期,G2/M期细胞减少,与阴性对照和空白对照组比较(P<0.01)差异有统计学意义,且野生型p53表达增加。结论:PPARγ基因静默能诱导HCCLM3细胞凋亡,抑制增殖;与促进野生型p53表达相关。
Objective ： To observe the effects of knocking down the expression of peroxisome proliferators-activated receptor gamma （PPARγ）with RNA interference techniques on the proliferation and apoptosis of hepatocellular carcinoma HCCLM3 cells. Methods：A short-hairpin RNA （shRNA） eukaryotic expression vector against PPARγwas constructed and transfected into HCCLM3 cells. The changes of PPARγ expression were detected by semi-quantitative RT-PCR and Western blotting analysis. The proliferation of HCCLM3 cells was tested by MTT assay. Apoptosis ratio of HCCLM3 cells was detected by TUNEL method and flow cytometry （FCM）. Expressions of PCNA and wide-type p53 protein were analyzed by immuocytochemistry （ICC） methods. Results：The sequence-specific shRNA （pshPPARγ）efficiently blocked the expression of PPARγ mRNA by 80.5%. At 48 h after transfection of pshPPARγ, proliferation of HCCLM3 cells was significantly suppressed by 71.5%. The positive rate of PCNA expression was （23.8± 7.2 ） % at 40 h transfection. The apoptotic rates were （24.2 ± 4.7 ） % as detected by TUNEL assay and （23.2 ±4.2） % of cells as measured by FCM test, respectively. The detection results of the two methods were consistent. In pshPPARγ transfection group, cell cycle of HCCLM3 cells was arrested in G0/G1 phase and the proportion of cells in G2/M phase decreased. Moreover, expression of wide-type p53 protein increased significantly. Conclusion ： Knockdown PPARγ expression with RNA interference technology can significantly suppress proliferation and induce apoptosis of HCCLM3 cells. It is related with up-regulation of wideotype p53 protein expression
出处
《肿瘤》
CAS
CSCD
北大核心
2008年第8期676-680,共5页
Tumor
