慢病毒介导shRNA干扰前列腺癌PC3 细胞Keap1基因表达的研究

中国临床药理学与治疗学 ›› 2014, Vol. 19 ›› Issue (8): 861-865.

慢病毒介导shRNA干扰前列腺癌PC3 细胞Keap1基因表达的研究

王天珩, 吴欣俐, 刘晓平

皖南医学院药学院,芜湖 241000,安徽

收稿日期:2014-04-27 修回日期:2014-07-29 出版日期:2014-08-26 发布日期:2014-08-26
通讯作者: 刘晓平,男,教授,研究方向:分子药理学。 E-mail: liuxiaoping@wnmc.edu.cn
作者简介:王天珩,女,硕士生,研究方向:分子药理学。 E-mail: 726719198@qq.com
基金资助:
国家自然科学基金项目(81272485)

Interference of shRNA on Keap1 in prostate cancer PC3 cell mediated by lentivirus

WANG Tan-heng, WU Xin-li, LIU Xiao-pin

Pharmacy School of Wannan Medical College, Wuhu 241000,Anhui,China

Received:2014-04-27 Revised:2014-07-29 Online:2014-08-26 Published:2014-08-26

摘要/Abstract

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Keap1的RNAi寡聚核苷酸序列;采用慢病毒载体构建Keap1的shRNA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Keap1靶向序列,以慢病毒载体构建完成对应Keap1的shRNA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Keap1的shRNA慢病毒载体,有效抑制PC3细胞中Keap1的表达。

关键词: Keap1, shRNA, 慢病毒, PC3细胞

Abstract: AIM: To construct the shRNA interference lentivirus vector for Keap1 and evaluate the interference effects in human prostate cancer PC3 cell mediated by lentivirus. METHODS: Bioinformatics methods were used to design RNAi sequences for Keap1. A lentiviral vector was applied for construction of Keap1 shRNA vectors, which was expressed in E. coli packaged by 293T cells. Transfection efficiency and titer were measured by dilution method according to the green fluorescent protein (GFP) tracer. Real-time fluorescence quantitative method was applied to compare interfere effects of target sequences.RESULTS: Four Keap1 targeting sequences were constructed completely and the corresponding Keap1 shRNA lentiviral vectors were screened for efficiency. One shRNA lentiviral vector with best efficiency was screened by transient transfection (interference efficiency reached 80%) and the working condition was established as well.CONCLUSION: shRNA lentiviral vectors for Keap1 was constructed and screened successfully in PC3 cells, which effectively inhibit the expression of Keap1.

Key words: Keap1, shRNA, lentivirus, PC3 cell

中图分类号:

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, 19(8): 861-865.

WANG Tan-heng, WU Xin-li, LIU Xiao-pin. Interference of shRNA on Keap1 in prostate cancer PC3 cell mediated by lentivirus[J]. Chinese Journal of Clinical Pharmacology and Therapeutics, 2014, 19(8): 861-865.

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