【摘要】 目的探讨细胞信号传导子和转录激活子6(STAT)信号传导通路与结肠癌细胞凋亡的关系。方法构建4个STAT6特异的shRNA质粒载体,应用阳离子脂质体瞬时转染人结肠癌细胞HT-29,以阻断Stat6信号通路;分别用逆转录聚合酶链反应(RT-PCR)方法检测STAT6mRNA表达和运用流式细胞术检测磷酸化STAT6蛋白(pSTAT6)表达来筛选有效的小发夹RNA(shRNA);运用流式细胞术分析细胞凋亡变化;RT-PCR和Western印迹观察Bcl-2和Bax基因表达。结果成功构建并筛选出两个STAT6特异的shRNA质粒载体;STAT6基因沉默的结肠癌细胞中凋亡细胞明显增加,空白对照组细胞早期凋亡率为0.4%,pshRNA-STAT6-1组和pshRNA-STAT6-4组分别为13.0%和8.8%(P<0.05);Stat6信号通路被阻断的结肠癌细胞中Bcl-2基因表达明显减少,而Bax蛋白基因表达明显增多(P<0.01)。结论Stat6信号传导通路能抑制结肠癌细胞凋亡,可能是通过调节Bcl-2和Bax基因的表达发挥作用。更多还原

【Abstract】 Objective To investigate the relationship between the signal transducer and activator of t ranscription 6 (STAT6) and human colon cancer.Methods Four STAT6-specific recombinant plasmid vectors, pshRNA-STAT6-1, 2 , 3, and 4 were constructed and transfected into the cultured human colon cancer cells of the line HT-29. Seventy-two hours later RT-PCR was used to detect t he mRNA expression of STAT6 and the apoptosis-related genes Bcl-2 and Bax, flo w cytometry (FCM) was used to detect the protein expression of phopho-STAT6 (pS TAT6). HT-29 cells were inoculated into a plate and transfected with pshRNA-ST AT6-1 or pshRNA-STAT6-4, and HT-29 cells without transfection were used as c ontrols. Seventy-two hours later FCM was used to observe the cell apoptosis. An other HT-29 cells were inoculated into a plate and transfected with pshRNA-STA T6-1 or pshRNA-STAT6-4, or blank liposome as controls. Seventy-two hours lat er. Western blotting was used to detect the protein expression of Bcl-2 and Bax genes. Results The p-STAT6 protein expression rate was 3.2%±0.6% in the pshRNA-ST AT6-1 group, significantly lower than that of the blank control group (18.2%±0 .9%, P<0.01) with an inhibition rate of 82.4%, and was 7.9%±0.4% in the psh RNA-STAT6-4 group, significantly lower than that in the blank control group to o (P<0.01) with an inhibition rate of 56.6%. And the p-STAT6 protein expres sion rates of the pshRNA-STAT6-2 and pshRNA-STAT6-3 groups were 16.6%±0.5% and 17.1%±0.7% respectively, both not significant different from that of the bl ank control group (both P>0.05). The early cell apoptosis rates of the pshRN A-STAT6-1 and pshRNA-STAT6-4 groups were 13.0% and 8.8% respectively, both s ignificantly higher than that of the blank control group (0.4%, both P<0.05) . The mRNA expression of Bcl-2 was significantly lower and the mRNA expression of Bax was significantly higher in the pshRNA-STAT6-1 and pshRNA-STAT6-4 gro ups than in the blank control and blank liposome groups (all P<0.01). The pr otein expression patterns of Bcl-2 and Bax was consistent with that of their pr otein expression. Conclusion STAT6 signaling pathway inhibits the apoptosis of colon cancer cells by regulation of the Bcl-2 and Bax genes.更多还原