NSBP1基因干扰RNA慢病毒载体的构建及效应检测  

Construction and efficacy identification of a lentiviral vector harboring RNAi based on gene NSBP1

作　　者：蒋宁[1] 周利群[1] 辛殿祺[1] 吕天敬[1] 韩文科[1] 

机构地区：[1]北京大学第一医院泌尿外科泌尿外科研究所,北京100034

出　　处：《基础医学与临床》2009年第12期1249-1253,共5页Basic and Clinical Medicine

基　　金：国家自然科学基金(30271295和30672099);北京市自然科学基金(7092101)

摘　　要：目的构建和鉴定NSBP1基因RNA干扰慢病毒表达载体。方法针对NSBP1mRNA设计了3条siRNA,并构建pGCSIL-GFP-NSBP1慢病毒质粒,测序鉴定。用pGCSIL-GFP-NSBP1、pHelper1.0和pHelper2.0质粒共转染293T细胞包装产生慢病毒,测定病毒滴度。将慢病毒转染前列腺癌DU145细胞,Western-blot检测NSBP1表达,MTT法检测细胞生长活性,用转染细胞种植裸鼠成瘤,观察抑瘤效果。结果PCR和测序证实,成功构建LV-shNSBP1的慢病毒载体,病毒滴度达2×108TU/mL。转染细胞中NSBP1蛋白表达显著降低,且MTT检测细胞生长活性明显减慢,转染细胞在裸鼠体内成瘤率没有影响,但对肿瘤的生长有明显抑制作用。结论人NSBP1基因RNA干扰慢病毒载体构建成功,且NSBP1对前列腺癌激素非依赖DU145细胞的生长具有重要作用。Objective To construct and identify the efficacy of a lentiviral vector harboring RNAi sequence targeting NSBP1 gene.Methods Three siRNA targeting the NSBP1 mRNA were designed,the pGCSIL-GFP-NSBP1 lentivirus vectors were constructed and confirmed by DNA sequencing.A total of 293T cells were co-transfected with pGCSIL-GFP-NSBP1,pHelper1.0 and pHelper2.0 for the virus stocks produced,the titer of the virus was tested.After lentivirus transfecting into DU145 cells,Western-blot and MTT methods were used to determine the expression and biological activity of NSBP1 gene,the cells were transplanted into nude mice,then inhibitive effect was observed.Results PCR analysis and DNA sequencing demonstrated that the RNAi sequence targeting the human NSBP1 gene was successfully inserted into the lentiviral vector.The titer of the recombinant lentiviral vector was 2×10^8TU/mL.NSBP1 protein expression level in transfected cells was significantly decreased and growth rate of cells transfected with lentivirus was decreased by MTT assay,the downregulation of NSBP1 reduced growth rate of transplantated tumor,whereas tumorgenicity was not influenced.Conclusion The construction of the lentiviral vector of NSBP1 has been successfully prepared and NSBP1 plays an important regulatory role in androgen-independent prostate cancer cell proliferation.

关 键 词：RNA干扰 核小体结合蛋白 慢病毒 WESTERNBLOT MTT 

分 类 号：Q78[生物学—分子生物学]