Bmi-1 shRNA慢病毒表达载体的构建及稳转U266细胞株的建立  

Establishment of a U266 Cell Line with Stable Bmi-1 Silencing by Lentivirus-mediated RNA Interference

作　　者：徐珍珍[1] 吴顺泉[1] 战榕[1] 

机构地区：[1]福建省血液病学重点实验室,福建省血液病研究所,福建医科大学附属协和医院

出　　处：《中国实验血液学杂志》2012年第2期473-477,共5页Journal of Experimental Hematology

基　　金：福建医科大学校苗圃基金资助(编号2010MP035);福建省血液病学重点实验室经费资助(编号2009J1004)

摘　　要：本研究构建Bmi-1基因的shRNA慢病毒表达载体,建立U266-li稳定细胞株,为下一步Bmi-1的功能研究及应用RNAi技术治疗打下基础。设计、合成1对针对Bmi-1 mRNA的shRNA序列,退火后连接到pLVTHM干扰载体上,与psPAX2、PMD2G共转染HEK 293T细胞,包装产生慢病毒颗粒并测定病毒滴度,感染U266细胞,建立稳定细胞株;应用实时PCR和Western blot技术分别检测U266稳定细胞中Bmi-1及P14 mRNA和蛋白水平的表达,并与对照组进行比较。结果表明,成功构建了针对Bmi-1基因的shRNA慢病毒表达载体,病毒滴度为5×107 TU/ml;建立稳定转染的U266细胞株。有效干扰验证显示,shBmi-1能明显降低Bmi-1的mRNA及蛋白水平,而P14的mRNA及蛋白水平则都上调,其差异有统计学意义(P<0.05)。结论:成功构建Bmi-1基因的shRNA慢病毒表达载体,建立稳定干扰Bmi-1表达的U266细胞株。This study was aimed to construct lentivirus-mediated shRNA expression vector targeting Bmi-1 and establish a stable cell line U266-li,so as to pave the way for further research on function of Bmi-1 and application of shRNA to gene therapy.One pair of oligonucleotide sequences targeted at human Bmi-1 mRNA were designed and synthesized.The annealed oligonucleotide fragments were subcloned into pLVTHM vector.Virus particles were collected after the control or shRNA vectors were co-transfected with the psPAX2 packaging plasmid and the plasmid pMD2.G was enveloped into HEK-293T cells by using Lipofectamine2000.The U266 cells were transdused with 5×106 recombinant lentivirus-transdusing units plus 6 μg/ml of polybrene.Real-time PCR and Western blot were used respectively to detect the expression of Bmi-1 and P14 after lentivirus transdusion.DNA sequencing demonstrated that the lentivirus RNAi vector of Bmi-1 was constructed successfuly and the virus was packaged in 293T cells.The titer of virus was 5 ×107 TU /ml.Stable transfected U266 cell line was established.As was expected,the mRNA and protein levels of Bmi-1 was reduced significantly in U266 cells after lentivirus transdusion,whereas the mRNA and protein levels of P14 was upregulated.It is concluded that the lentiviral RNAi vector of Bmi-1 is constructed,and U266 stable cell line is established.

关 键 词：BMI-1基因 RNA干扰 U266细胞 

分 类 号：R733.3[医药卫生—肿瘤] Q782[医药卫生—临床医学]