shRNA-Bmi1重组载体构建及其对胆囊癌裸鼠移植瘤hTERT表达的影响  

Construction of ShRNA-Bmil Recombinant Vector and Its Effect on The Expression of hTERT in Transplantated Tumor of Nude Mice with Gallbladder Carcinoma

作　　者：王琨[1] 魏东[1] 邹浩[1] 戈佳云[1] 李晓[1] 王琳[1] 张小文[1] 

机构地区：[1]昆明医科大学第二附属医院肝胆胰外科,云南昆明650101

出　　处：《昆明医科大学学报》2016年第4期8-14,共7页Journal of Kunming Medical University

基　　金：云南省应用基础研究计划项目(2011FZ124)

摘　　要：目的体外构建及鉴定sh RNA-Bmi1重组载体,探讨靶向沉默原癌基因Bmi1对人胆囊癌细胞Bmi1m RNA表达、蛋白表达及对裸鼠皮下移植瘤h TERT表达的影响.方法针对Bmi1不同位点构建4个sh RNABmi1重组质粒,采用倒置荧光显微镜观察转染效率,RT-PCR和Western blot检测各组Bmi1m RNA和蛋白表达,选择有效干扰组进行体内实验.构建GBC-SD裸鼠皮下移植瘤模型,随机分为sh RNA-Bmi-4组(实验组)、sh RNA-Scramble(错配组),Lipofectamine(阴性对照组),GBC-SD(空白对照组).成瘤后进行移植瘤治疗,治疗6周后处死裸鼠,免疫组化检测裸鼠移植瘤h TERT蛋白表达水平.结果成功构建了sh RNA-Bmi1重组载体.转染胆囊癌48 h后倒置荧光显微镜观察发现sh RNA-Bmi1-4组GBC-SD绿色荧光强度增强、转染效率最高,RT-PCR及Western blot结果显示sh RNA-Bmi1-4组m RNA及蛋白表达量均明显低于各对照组(P<0.05),对照组间表达量差异无统计学意义(P>0.05).选取sh RNA-Bmi1-4组作为有效干扰序列作后续体内实验.免疫组化检测sh RNA-Bmi-4组裸鼠移植瘤h TERT蛋白表达水平明显低于各对照组(P<0.05),对照组间蛋白表达量差异无统计学意义(P>0.05).结论体外成功构建了sh RNA-Bmi1重组载体,顺利进行重组载体的转化和转染实验。靶向沉默Bmi1基因可有效促进胆囊癌细胞Bmi1m RNA降解、抑制其蛋白的表达,同时有效抑制GBC-SD裸鼠移植瘤h TERT蛋白表达.Objective To construct and identify sh RNA- Bmi1 recombinant vector in vitro, and to explore the effects of target silencing proto oncogene Bmi1 on the Bmi1 m RNA and protein expression in human gallbladder carcinoma cell and the expression of h TERT on subcutaneously transplantated tumor of human gallbladder carcinoma in BALB/c Nude Mice. Me thods Four sh RNABmi1 recombinant plasmids were successfully constructed according to different Bmi1 sites. Transfection efficiency was observed by inverted fluorescence microscope and RT- PCR and Western blot were used to measure m RNA and protein expression of Bmi- 1 in gallbladder cancer cells. The effective interference group was selected for the vivo experiment. The subcutaneous transplantation tumor model of human GBC- SD cell line was successfully constructed in BALB/c nude Mice and randomly divided into sh RNA- Bmi- 4group（experimental group）, sh RNA- Scramble（mismatch group）, Lipofectamine（negative control group）,GBC- SD（blank control group）. After established the subcutaneous transplantation tumor model, the nude mice were killed after the treatment for 6 weeks. The expression level of h TERT protein in nude mice transplanted tumor was detected by immunohistochemistry. Re s ults sh RNA- Bmi1 recombinant vector was successfully constructed. The most effective interferencing plasmids were transfected into GBC- SD cells for 48 hour. The highest green fluorescence intensity and transfection efficiency of sh RNA- Bmi1- 4 group GBC- SD were observed by inverted fluorescence microscope. RT- PCR and Western blot results showed that the expression levels of Bmi1 m RNA and protein in sh RNA- Bmi1- 4 group were significantly lower than that in the control groups（P 0.05）, and there was no significant difference between control groups（P 0.05）.The sh RNA- Bmi1- 4 group was selected as the effective interference sequence for the following in vivo experiments. The expression level of h TERT protein on transplantation tumor in nude mice in sh RNA- Bmi- 4 gro

关 键 词：shRNA-Bmi1 重组载体 GBC-SD 皮下移植瘤 hTERT 

分 类 号：R735.8[医药卫生—肿瘤]