RNA干扰沉默IGF1R表达对A549细胞凋亡及化疗敏感性的观察  

Effect of silence IGF1R by shRNA on apoptosis and chemotherapysensitivity of A549 cells

作　　者：杜永亮[1] 赵杰[1] 贾晓民[1] 李海泉[1] 王海清[1] 徐俊马[1] 

机构地区：[1]徐州医学院第二附属医院呼吸科,江苏徐州221006

出　　处：《中华肿瘤防治杂志》2013年第11期835-839,共5页Chinese Journal of Cancer Prevention and Treatment

基　　金：江苏省徐州市科技计划(XF11C102)

摘　　要：目的:shRNA重组慢病毒感染A549细胞沉默IGF1R基因,观察其对A549细胞凋亡及紫杉醇化疗敏感性的影响。方法:构建IGF1R-shRNA-LV重组慢病毒,并以MOI=20感染A549细胞,评价感染效果;应用RT-PCR和蛋白质印迹法检测IGF1R干扰效果;蛋白质印迹检测pro-Caspase-3、cleaved-Caspase-3、Fas和FasL蛋白相对表达量,Hoechst33258法检测细胞凋亡形态学变化;CCK-8法检测细胞对紫杉醇敏感性变化。结果:功构建IGF1R-shRNA真核表达质粒,并包装出重组慢病毒,对照组和实验组病毒滴度分别为(1.9E+9T)和(7.0E+8T)U/mL,A549感染效率>90%;空白对照组、阴性对照组和实验组比较,A549细胞IGF1R mRNA相对表达量分别为1.55±0.09、1.53±0.18和0.39±0.02,实验组显著下降,沉默效率为74.51%,t=11.145,P=0.000;IGF1R蛋白相对表达量分别为0.97±0.06、0.95±0.08和0.28±0.01,沉默效率为70.53%,t=33.147,P=0.000;pro-Caspase-3蛋白相对表达量分别为0.62±0.02、0.64±0.02和0.35±0.04,t=5.536,P=0.006;cleaved-Caspase-3蛋白相对表达量分别为0.07±0.01、0.08±0.01和0.16±0.02,表明Caspase-3被大量激活、分解,促进细胞凋亡,t=-3.438,P=0.025;Fas蛋白相对表达量分别为0.41±0.06、0.43±0.07和0.88±0.03,有利于启动死亡受体凋亡途径,t=-5.756,P=0.005;Fasl蛋白相对表达量分别为0.20±0.02、0.20±0.03和0.28±0.04,组间比较差异无统计学意义,t=-1.239,P=0.283。Hoechst33258染色见实验组细胞核深染增加,凋亡细胞显著增多。空白对照组、阴性对照组和实验组紫杉醇IC50分别为55.08、54.61和18.70ng/mL,紫杉醇敏感性显著增加。结论:构建的重组慢病毒能有效抑制IGF1R表达,诱导A549细胞凋亡,提高A549细胞对紫杉醇敏感性。OBJECTIVE：To study effect of silencing IGFIR by shRNA lentiviral vector on apoptosis and sensitivity to paclitaxel of A549 Cells. METHODS： ShRNA lentiviral vectors targeting IGF1R gene were constructed and transfected to A549 cells by MOI：20. The infection effect was evaluated on the basis of fluorescent protein GFP. Efficiency of silen- cing IGF1R was detected by real time PCR and Western blot. The protein expression of pro-Caspase-3, cleaved-Caspase-3, Fas and FasL was measured by Western blot. The cell apoptosis was detected by Hoechst33258 and the sensitivity of A549 cells to paclitaxel was detected by CCK-8 method. RESULTS： The IGF1R-shRNA-LV recombinant lentiviral was constructed successfully. Before and after A549 cells infectioned, IGFIR relative expressions at the levels of mRNA and protein were 1. 55±0.09,1.53±0.18,0.39 ±0.02 （t= 11. 145,P=0. 000） and 0. 97 ± 0. 06 , 0. 95 ±0.08,0.28±0.01 （t：33. 147,P=0. 000）. The efficiency of silencing IGFIR was 74.5% and 70.53% at the level of mRNA and protein. The protein expression of pro-Caspase-3 was 0.62 ± 0.02, 0.64± 0.02, 0.35 ±0.04 （t ： 5. 536, P ： 0. 006） and cleaved-Caspase-3 was 0. 07±0. 01,0. 08±0. 01, 0.16±0. 02 （t=3. 438,P=0. 025） and Fas was 0.41±0.06, 0.43±0.07.0.88±0.03 （t=--5.756,P：0.005） and FasL was 0.20±0.02,0.20±0.03,0.28±0.04 （t=-l.239,P： 0. 283）,Apoptotic cells were increased significantly by Hoechst33258. ICs0 of paclitaxel to A549 cells declined from 55.08 ng/mL,54.61 ng/mL to 18.70 ng/mL. CONCLUSION：The recombinants could down-regulate the expression of lGF1R.induce aDoDtosis and enhance the sensitivity of A549 cells to paclitaxel.

关 键 词：慢病毒 短发卡RNA 人胰岛素样生长因子I类受体 A549细胞 紫杉醇 细胞凋亡 

分 类 号：R73-3[医药卫生—肿瘤]