MUC1黏蛋白启动子的克隆及在胰腺癌Panc-1细胞中的转录活性  

Cloning and analyzing the activity of the promoter of the MUC1 gene in Panc-1 cells

作　　者：潘耀振[1] 孙诚谊[1] 詹磊[1] 张浩[1] 田舍[1] 张宏[1] 姜楠[1] 

机构地区：[1]贵阳医学院附属医院肝胆外科,550004

出　　处：《中华肝胆外科杂志》2014年第2期137-141,共5页Chinese Journal of Hepatobiliary Surgery

基　　金：贵州省肝胰疾病研究科技创新人才团队基金资助项目（黔科合人才团队[2010]4010）

摘　　要：目的 克隆MUC1黏蛋白启动子,研究MUC1启动子在人类胰腺癌细胞株Panc-1细胞和人类宫颈癌细胞株Hela细胞中的转录活性.方法 采用巢式PCR扩增MUC1启动子片段并酶切连接至含有EGFP报告基因的pEGFP-N1载体中构建质粒pEGFP-MUC1-N1,采用基因重组方法将MUC1启动子片段及EGFP报告基因构建于pShuttle质粒上形成pShuttle-MUC1-EGFP质粒.通过脂质体共转染人胰腺癌细胞株Panc-1和人宫颈癌细胞株Hela.使用荧光素酶检测系统（Luciferase assay system）测定细胞的荧光素酶活性,通过荧光显微镜和流式细胞仪检测MUC1启动子在Panc-1细胞中的特异转录活性.结果 成功克隆出MUC1启动子,双酶切、PCR检测和DNA测序证实pEGFPMUC1-N1、pShuttle-MUC1-EGFP载体构建成功.重组报告载体荧光素酶活性显著升高（t=18.975,P =0.001）.经pEGFPMUC1-N1、pShuttleMUC 1-EGFP质粒转染后MUC1启动子在Panc-1细胞中的转录活性为阳性对照CMV启动子活性的69.6％及63.6％,明显高于Hela细胞中的4.2％及3.7％,在胰腺癌细胞中具有较高特异性,且在胰腺癌细胞中的活性明显高于阴性对照pGL3-Basic的0.093％.结论 MUC1启动子在淋巴瘤细胞中具有较高的特异性,可以作为胰腺癌细胞基因转染的肿瘤特异性启动子使用.此研究为进一步运用MUC1启动子在基因水平靶向治疗胰腺癌奠定了基础.Objective To evaluate the MUC1 promoter＇s role in driving gene expression in pancreatic cancer and its therapeutic significance.Methods Two plasmids were made.The plasmid pEGFP-MUC1N1 contained MUC1 promoter fragment connected to the pEGFP-N1 vector with the EGFG reporter gene.The pShuttle-MUC1-EGFP plasmid contained MUC1 promoter fragment and EGFP reporter gene connected to pShuttle plasmid.Lipofectamine 2000 was used to transfect the two plasmids into cells of MUC1-positive human pancreatic cell line Panc-1 and MUC1-negative human cervical carcinoma Hela.Fluorescence microscopy and flow cytometry compared the specificity and activity of the MUC1 promoter and CMV promoter.Results Reporter gene EGFP-positive cells 48 hours after transfection with pEGFP-MUC1-N1 and pShuttleMUC1-EGFP plasmid were 69.6% and 63.6% respectively,in Panc-1 cells,and 4.2% and 3.7% respectively,in Hela cells.Conclusions MUC1 promoter can drive reporter gene activity in MUC1-positive tumor cells targeting functional expression.There is potentially a use of targeted therapy in pancreatic cancer at the genetic level.

关 键 词：胰腺癌 MUC1启动子 双荧光素酶活性检测 

分 类 号：R735.9[医药卫生—肿瘤]