慢病毒介导siRNA沉默EGFR基因及其对胰腺癌细胞增殖和凋亡的影响  

Effect of EGFR gene inhibition by lentivirus-mediated small interfering RNA on the proliferation and apoptosis of human pancreatic cancer cells

作　　者：法镇中[1] 江勇[2] 谭玉林[1] 吴宝强[2] 石亮荣[3] 

机构地区：[1]常州市武进人民医院普外科,江苏213003 [2]苏州大学附属第三医院肝胆外科 [3]苏州大学附属第三医院肿瘤内科

出　　处：《中华肝脏外科手术学电子杂志》2013年第2期39-43,共5页Chinese Journal of Hepatic Surgery(Electronic Edition)

基　　金：国家青年科学基金项目(81201741);江苏省卫生厅面上科研课题(H200870);常州市卫生局重大招标项目(ZD200811);江苏省常州市社会发展项目(CS2008912)

摘　　要：目的探讨利用慢病毒介导小干扰核糖核酸(siRNA)沉默表皮生长因子受体(EGFR)基因的可行性及其对胰腺癌细胞增殖和凋亡的影响。方法构建慢病毒表达载体LV-shEGFR,包装慢病毒表达载体及制备慢病毒颗粒EGFR-shRNA。实验分为Panc-1组(空白组)、绿色荧光蛋白(GFP)-Panc-1组(对照组)及siEGFR-Panc-1组(干扰组)共3组。空白组使用未经处理的对数生长期的胰腺癌细胞株Panc-1细胞,对照组使用不含siRNA片段的慢病毒颗粒及干扰组使用制备好的EGFR-shRNA慢病毒颗粒感染对数生长期的Panc-1细胞。采用实时聚合酶链反应(real-time PCR)检测EGFR基因表达量(以与β-肌动蛋白相对浓度比值表示),采用蛋白质印迹法(Western-blot)检测细胞中EGFR蛋白表达(以灰度值表示),采用噻唑蓝(MTT)比色法检测细胞增殖情况,采用流式细胞仪检测细胞周期及细胞凋亡情况。3组EGFR表达、吸光度(A)值、细胞凋亡率、细胞周期比较采用单因素方差分析和t检验。结果干扰组EGFR基因相对含量为0.20±0.04,明显低于对照组(1.00±0.15)及空白组(1.13±0.13),差异有统计学意义(LSD-t=7.865,7.668;P<0.05)。干扰组EGFR蛋白表达量为0.185±0.009,与对照组(0.801±0.087)及空白组(0.825±0.092)相比,干扰组EGFR蛋白表达明显降低(LSD-t=4.216,3.975;P<0.05)。干扰组细胞增殖受抑制,生长速度较对照组生长缓慢。干扰组G2/M期细胞比率为(8.87±0.29)%,与空白组(20.00±1.88)%和对照组(21.48±2.13)%比较明显减少,比较差异有统计学意义(LSD-t=2.015,2.323;P<0.05),干扰组S期细胞比率为(50.97±3.04)%,与空白组(36.67±6.18)%和对照组(39.91±2.09)%比较明显增加,比较差异有统计学意义(LSD-t=1.987,2.251;P<0.05)。干扰组细胞凋亡率为(18.4±2.3)%明显高于对照组(7.4±1.4)%和空白组(7.7±1.2)%,比较差异有统计学意义(LSD-t=2.585,2.667;P<0.05)。结论慢病毒介导siRNA可以沉默EGFR基因,抑制胰腺癌细胞增殖,阻滞细胞在S期Objective To explore the feasibility of lentivirus-mediated small interfering RNA （siRNA） silencing the epidermal growth factor receptor （EGFR）and its influence on the proliferation and apoptosis of human pancreatic cancer cell. Methods The expression vector LV-shEGFR was established and packaged and the lentivirus particles containing EGFR-shRNA was collected for use. The experiment was divided into Panc-1 group （blank group）, green fluorescent protein （GFP）-Panc-1 group （control group） and siEGFR-Panc-1 group （interference group）. The pancreatic cancer cell line Panc-1 cells at logarithmic growth phase in the blank group received no treatment and the cells in the control group were infected by lentivirus particles without siRNA fi＇agment. For the interfering group, the Panc-1 cells were infected with the prepared lentiviral particles containing EGFR-shRNA. The expression of EGFR gene （described as relative ratio with β-action） and protein （described as gray value） were examined by real-time polymerase chain reaction （real-time PCR）and Western-blot respectively. The cell proliferation, as well as cell cycle and apoptosis were detected by methyl thiazolyl tetrazolium （MTT） assay and flow cytometry respectively. The EGFR expression, absorbance （A） value, apoptosis rate and cell cycle in three groups Were compared using one-way analysis of variance （ANOVA） and t-test. Results The relative expression level of EGFR gene in the interference group was 0.20±0.04, which was significantly lower compared with the control group （1.00±0.15） and the blank group （1.13±0.13）. There were significant differences （LSD-t=7.865, 7.668; P〈0.05）. The EGFR protein expression level in the interference group was 0.185±0.009, which was down-regulated compared with the control group （0.801±0.087） and the blank group （0.825±0.092） （LSD-t =4.216,3.975; P〈0.05）. The eell proliferation of the interference group was obviously suppressed, which was slower compar

关 键 词：癌 胰腺管 受体 表皮生长因子 慢病毒 小干扰核糖核酸 细胞增殖 细胞凋亡 细胞周期 

分 类 号：R735.9[医药卫生—肿瘤]