siRNA干扰HepG2.2.15细胞BUBR1基因及对细胞增殖的影响  

siRNA interference of BubR1 gene in HepG2.2.15 cells and its effect on cell proliferation

作　　者：周艳萌 向晓波 王欢 Zhou Yanmeng;Xiang Xiaobo;Wang Huan(Department of Microbiology,School of Basic Medicine,Zunyi Medical University,Zunyi Guizhou 563099,China;Stomatological Hospital Affiliated of Zunyi Medical University,Zunyi Guizhou 563099,China)

机构地区：[1]遵义医科大学基础医学院微生物学教研室,贵州遵义563099 [2]遵义医科大学附属口腔医院,贵州遵义563099

出　　处：《遵义医科大学学报》2020年第6期703-708,共6页Journal of Zunyi Medical University

基　　金：贵州省教育厅自然科学基金资助项目(NO:黔教科2010041)。

摘　　要：目的利用慢病毒载体和RNAi干扰技术沉默稳定转染了HBV全基因组的肝癌细胞HepG2.2.15的BUBR1基因,并检测沉默后对细胞增殖的影响。方法设计BUBR1基因的小干扰RNA,构建稳定遗传的慢病毒载体质粒,并进行测序鉴定。测序正确后用293T细胞进行包装并获得具有高感染性的BUBR1 shRNA重组慢病毒,用重组慢病毒感染HepG2.2.15细胞,通过荧光显微镜观察绿色荧光蛋白的表达情况;Real-time PCR检测沉默效果;MTT法检测沉默BUBR1后对HepG2.2.15细胞增殖的影响。结果测序结果证明干扰载体中的特异性干扰序列完全正确,测序鉴定正确的BUBR1 shRNA重组慢病毒转染293T细胞48 h,荧光显微镜下可见绿色荧光,病毒滴度约为10-9 TU/mL;制备成功的重组慢病毒BUBR1 shRNA以MOI=10感染HepG2.2.15细胞,72 h后转染率均达80%以上,用嘌呤霉素筛选得到稳转细胞株;Real-time PCR检测显示3对shRNA靶点转染HepG2.2.15细胞后,均显著下调BUBR1 mRNA的转录水平;MTT结果显示沉默BUBR1对HepG2.2.15细胞增殖没有明显影响。结论成功构建BUBR1基因慢病毒干扰载体,该病毒载体能有效沉默HepG2.2.15细胞的BUBR1基因,沉默后不影响HepG2.2.15细胞的增殖。Objective Using lentiviral vector and RNAi interference technology to silence the BubR1 gene of HepG2.2.15 cell line.which is a hepatoma cell stably transfected with HBV genome,to detect the effect of silencing on cell proliferation.Methods The small interfering RNA(siRNA)of BubR1 gene was designed to construct a stable lentiviral vector plasmid and sequenced.after the sequencing was correct,the recombinant lentivirus was packaged with 293T cells and highly infectious BubR1 shRNA was obtained.HepG2.2.15 cells were infected with recombinant lentivirus.The expression of green fluorescent protein was observed by fluorescence microscope.Real time PCR was used to detect the silencing effect,and MTT method was used to detect the effect on HepG2.2.15 cell proliferation.Results Sequencing results showed that the specific interference sequence in the interference vector was completely correct.Sequencing identified that the correct BubR1 shRNA recombinant lentivirus was transfected into 293T cells for 48 hours.The green fluorescence was observed under the fluorescence microscope,and the virus titer was about 10-9 TU/ml.HepG2.2.15 cells were infected with the recombinant lentivirus BubR1 shRNA with MOI=10.After 72 hours,the transfection rate was more than 80%.The stable cell lines were screened by puromycin.Real time PCR analysis showed that the three shRNA targets in transfected HepG2.2.15 cells significantly down regulated the transcription level of BubR1 mRNA;MTT results showed that silencing BubR1 had no significant effect on the proliferation of HepG2.2.15 cells.Conclusion BubR1 gene lentiviral interference vector was successfully constructed.The virus vector could effectively silence the BubR1 gene of HepG2.2.15 cells without affecting the proliferation of HepG2.2.15 cells.

关 键 词：BUBR1 RNA干扰 HEPG2.2.15细胞 慢病毒 细胞增殖 

分 类 号：R373[医药卫生—病原生物学]