| 摘要: |
| ［摘要］目的 体外构建及鉴定shRNA-Bmi1重组载体，探讨靶向沉默原癌基因Bmi1对人胆囊癌细胞Bmi1mRNA表达、蛋白表达及对裸鼠皮下移植瘤hTERT表达的影响．方法 针对Bmi1不同位点构建4个shRNABmi1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Bmi1mRNA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shRNA-Bmi-4组（实验组）、shRNA-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hTERT蛋白表达水平．结果 成功构建了shRNA-Bmi1重组载体．转染胆囊癌48 h后倒置荧光显微镜观察发现shRNA-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shRNA-Bmi1-4组mRNA及蛋白表达量均明显低于各对照组（P＜0.05），对照组间表达量无显著差异（P＞0.05）．选取shRNA-Bmi1-4组作为有效干扰序列作后续体内实验．免疫组化检测shRNA-Bmi-4组裸鼠移植瘤hTERT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无显著差异（P＞0.05）．结论 体外成功构建了shRNA-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Bmi1mRNA降解、抑制其蛋白的表达，同时有效抑制GBC-SD裸鼠移植瘤hTERT蛋白表达. |
| 关键词: ［关键词］shRNA-Bmi1 重组载体 GBC-SD 皮下移植瘤 hTERT |
| DOI： |
| 分类号: |
| 基金项目:［基金项目］云南省应用基础研究计划项目（2011FZ124） |
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| Construction of ShRNA-Bmi1 Recombinant Vector and Its Effect on The Expression of hTERT in Transplantated Tumor of Nude Mice with Gallbladder Carcinoma |
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WANG Kun
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（Dept. of Hepatopancreatobiliary Surgery，The Second Affiliated Hospital of Kunming Medical University， Kunming 650101，China ）
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| Abstract: |
| ［Abstract］Objective To construct and identify shRNA-Bmi1 recombinant vector in vitro， and to explore the effects of target silencing proto oncogene Bmi1 on the Bmi1mRNA and protein expression in human gallbladder carcinoma cell and the expression of hTERT on subcutaneously transplantated tumor of human gallbladder carcinoma in BALB/c Nude Mice. Methods Four shRNABmi1 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 mRNA 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 shRNA-Bmi-4 group（experimental group），shRNA-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 hTERT protein in nude mice transplanted tumor was detected by immunohistochemistry. Results shRNA-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 shRNA-Bmi1-4 group GBC-SD were observed by inverted fluorescence microscope. RT-PCR and Western blot results showed that the expression levels of Bmi1mRNA and protein in shRNA-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 shRNA-Bmi1-4 group was selected as the effective interference sequence for the following in vivo experiments. The expression level of hTERT protein on transplantation tumor in nude mice in shRNA-Bmi-4 group was significantly lower than that in control group（P < 0.05）， and there was no significant difference between the control groups（P > 0.05）. Conclusions shRNA-Bmi1 recombinant vector has been successfully constructed in vitro， and the transformation and transfection experiments of the recombinant vector have been carried out successfully. Targeted silencing Bmi1 gene can effectively promote Bmi1mRNA degradation，inhibit the Bmil protein expression in the GBC-SD cell line and the expression of hTERT protein in subcutaneously transplantated tumor of GBC-SD in Nude Mice. |
| Key words: ［Key words］shRNA-Bmi1 Recombinant vector GBC-SD Subcutaneously transplantated tumor hTERT |