用流式细胞仪进行人核迁移蛋白shRNA有效干扰片段的分析和筛选  

Analysis and Selection of Human Nuclear Distribution Protein C-specific shRNA by Flow Cytometry

作　　者：廖霞[1] 邹宇光[1] 庞实锋[1] 郑克勤[1] 周汝滨[1] 曹定国[1] 梁朋[1] 胡传银[1] 

机构地区：[1]广东医学院基础医学院生物学教研室,广东湛江524023

出　　处：《南昌大学学报（医学版）》2012年第8期1-5,9,共6页Journal of Nanchang University：Medical Sciences

基　　金：教育部科学技术研究重点项目(210156);广东省卫生厅青年基金(B2010235);广东医学院博士启动基金(XB1007);广东省大学生创新实验项目(KY1003);湛江市科技攻关项目(2010C3111005)

摘　　要：目的为了更好地利用流式细胞仪筛选出有效的RNA干扰片段,构建了一系列真核表达载体pDs-NUDC-RFP-TK-U6-shNUDC-A、B、C、D、E、F、H。方法将NUDC-RFP融合基因克隆至pDs载体中,构建出pDs-NUDC-RFP.载体;将人U6启动子克隆至pDs-NUDC-RFP.载体中;将shNUDC-A、B、C、D、E、F、H各个片段分别克隆至含有U6启动子的载体中,形成一系列RNA干扰载体pDs-NUDC-RFP-TK-U6-shNUDC-A、B、C、D、E、F、H;将提取高质量的质粒通过脂质体方法转染293T细胞。用流式细胞仪分析转染的293T细胞的荧光强度和荧光细胞数量,用实时荧光相对定量RT-PCR(real-time PCR)检测NUDC-RFP mRNA的转录情况,用Western Blot法检测NUDC-RFP融合蛋白的表达水平。结果流式细胞仪检测结果显示:空白对照组的293T细胞无发光细胞;阴性对照组的293T细胞能正常发光,且发光细胞数量较多;实验组的293T细胞发光细胞数量少,荧光强度低,说明其能有效干扰NUDC-RFP融合基因的表达,pDs-NUDC-RFP-TK-U6-shNUDC-F干扰下调效果约90%左右;pDs-NUDC-RFP-TK-U6-shNUDC-A可干扰hNUDC-RFP融合基因的表达,下调效果约85%左右;其他干扰片段pDs-NUDC-RFP-TK-U6-shNUDC-B、C、D、E、H下调效果在62%～75%;阴性对照组对融合基因无干扰效果;流式细胞仪检测结果与real-time PCR检测结果一致,也与Western Blot法的鉴定结果基本符合,并确定了shNUDC-F为RNA干扰效果最佳的干扰片段。结论流式细胞仪分析方法是一种有效的带荧光标记的RNA干扰载体的筛选方法,为后期研究打下了基础。Objective To select the effective human nuclear distribution protein C（hNUDC）-specific shRNA using flow cytometry through constructing vectors pDs-NUDC-RFP-TK-U6-shNUDC-A,B,C,D,E,F and H.Methods NUDC-RFP fused gene was cloned into pDs vector,resulting in pDs-NUDC-RFP.Human U6 promoter and hNUDC-specific shRNA were cloned into pDs-NUDC-RFP vector,resulting in pDs-NUDC-RFP-TK-U6-shNUDC-A,B,C,D,E,F and H.Then 293T cells were transfected with high-quality plasmid by liposome complex method.The fluorescence intensity and the number of fluorescent cells were detected by flow cytometry.NUDC-RFP mRNA was analyzed by quantitative real-time PCR and NUDC-RFP protein was detected by Western Blot.Results The 293T cells in blank control group did not show RFP fluorescence.However,a large number of cells in negative control group showed strong expression of RFP,but the fluorescence intensity and the number of fluorescent cells were significantly reduced in 293T cells transfected with hNUDC-specific shRNAs compared with negative control group.The levels of NUDC-RFP fusion protein expression were decreased by 90% and 85% in cells transfected with shNUDC-F and cells transfected with shNUDC-A,respectively.In addition,NUDC-RFP expression was down-regulated by 62%-75% by transfection with pDs-NUDC-RFP-TK-U6-shNUDC-B,C,D,E and H,respectively.NUDC-RFP gene expression was not disrupted in negative control group.There results were consistent with real-time RT-PCR and Western Blot analysis and showed that shNUDC-F was the best effective interference fragment.Conclusion Flow cytometry analysis is an effective method for selecting shRNA vector with fluorescent marker.

关 键 词：人核迁移蛋白 红色荧光蛋白 融合基因 流式细胞仪 

分 类 号：R393[医药卫生—基础医学] Q782[生物学—分子生物学]