慢病毒介导shRNA干扰前列腺癌PC3细胞Keap1基因表达的研究 |
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引用本文: | 王天珩,吴欣俐,刘晓平.慢病毒介导shRNA干扰前列腺癌PC3细胞Keap1基因表达的研究[J].中国临床药理学与治疗学,2014(8):861-865. |
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作者姓名: | 王天珩 吴欣俐 刘晓平 |
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作者单位: | 皖南医学院药学院,安徽芜湖241000 |
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基金项目: | 国家自然科学基金项目(81272485) |
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摘 要: | 目的:针对Keap1构建shRNA慢病毒干扰载体,并评价慢病毒介导的RNA干扰在人前列腺癌细胞PC3中的基因沉默效应。方法:利用生物信息学方法设计针对Keapl的RNAi寡聚核苷酸序列;采用慢病毒载体构建Keapl的shR-NA载体,利用大肠杆菌进行重组表达,利用293T细胞包装得到重组腺病毒;依据绿色荧光蛋白(GFP)示踪,逐孔稀释法确定转染效率及滴度;以实时荧光定量法比较各靶序列的基因干扰效果。结果:筛选了所构建的4个Keapl靶向序列,以慢病毒载体构建完成对应Keapl的shR-NA质粒。通过瞬时转染筛选得到效率最佳(干扰效率达到80%)的靶序列和工作条件。结论:本研究成功构建并筛选了针对Keapl的shRNA慢病毒载体,有效抑制PC3细胞中Keapl的表达。
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关 键 词: | Keapl shRNA 慢病毒 PC3细胞 |
Interference of shRNA on Keapl in prostate cancer PC3 cell mediated by lentivirus |
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Institution: | WANG Tan-heng, WU Xin-li, LIU Xiao-pin Pharmacy School of Wannan Medical College, Wuhu 241000 ,Anhui,China |
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Abstract: | AIM: To construct the shRNA in- terference lentivirus vector for Keapl and evalu- ate the interference effects in human prostate cancer PC3 cell mediated by lentivirus. METH- ODS: Bioinformatics methods were used to de- sign RNAi sequences for Keapl. A lentiviral vector was applied for construction of Keapl shRNA vectors, which was expressed in E. coli packaged by 293T cells. Transfection efficiency and titer were measured by dilution method ac- cording to the green fluorescent protein (GFP) tracer. Real-time fluorescence quantitative method was applied to compare interfere effects of target sequences. RESULTS: Four Keapl tar-geting sequences were constructed completely and the corresponding Keapl shRNA lentiviral vectors were screened for efficiency. One shR- NA lentiviral vector with best efficiency was screened by transient transfection (interference efficiency reached 80 %) and the working condi- tion was established as well. CONCLUSION. shRNA lentiviral vectors for Keapl was con- structed and screened successfully in PC3 cells, which effectively inhibit the expression of Keapl. |
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Keywords: | Keapl shRNA lentivirus PC3cell |
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