摘要
[目的]比较CMVE-PEG3p、PEG-3启动子在DU145细胞中的启动活性,为前列腺癌靶向性基因治疗提供依据。[方法]用PCR法扩增CMV增强子、PEG-3启动子;在真核表达质粒pShuttle-EGFP基础上分别构建2种启动子调控的、以绿色荧光蛋白为目的基因的真核表达质粒pShuttle-CMVE-PEG3p-EGFP、pShuttle-PEG3p-EGFP。将重组质粒用脂质体分别转染前列腺癌DU145细胞和人正常前列腺上皮细胞RWPE-1,72h后用Imagepro-Plus6.0分析2种启动子在相同时间内启动绿色荧光蛋白的表达水平。[结果]重组质粒在DU145细胞中观察到了绿色荧光,在RWPE-1细胞中无绿色荧光。pShuttle-CMVE-PEG3p-EGFP质粒在DU145细胞中表达强于pShuttle-PEG3p-EGFP,2种质粒在DU145细胞中的荧光强度(IOD)分别为246.22、130.93。[结论]所克隆的CMVE-PEG3p启动子和PEG-3启动子在前列腺癌DU145细胞中均表现出肿瘤特异性,其中CMVE-PEG3p启动子具有更强的启动效应,有望开发成为前列腺癌靶向性基因治疗的工具。
[Purpose] To compare the transcriptional activities of tumor-specific CMVE-PEG3 promoter and PEG-3 promoter in human prostate cancer DU145 cells,to provide base for targeting gene therapy in human prostate cancer.[Methods] The fragment of CMVE-PEG3 promoter and PEG-3 promoter were amplified by PCR.The eukaryotic expression plasmids （pShuttle-CMVE-PEG3p-EGFP and pShuttle-PEG3p-EGFP）which have enhanced green fluorescence protein reporter gene and modulated by CMVE-PEG3 promoter and PEG-3 promoter respectively were constructed based on the pShuttle-EGFP.These recombinant plasmids were transfected into DU145 and RWPE-1 cells with liposome.After 72 hours, the expression level of green fluorescent target protein modulated by 2 promoters at the same time was analyzed by Imagepro-Plus6.0 software.[Results] The green fluorescent target protein was observed in transfected DU145 cells,but there was no green fluorescence in transfected RWPE-1 control cells.The pShuttle-CMVE-PEG3p-EGFP had higher expression activity than pShuttle-PEG3p-EGFP in DU145 cells and the strength rate （IOD） of green fluorescence of DU145 cells transfected by pShuttle-CMVE-PEG3p-EGFP and pShuttle-PEG3p-EGFP were 246.22 and 130.93 respectively.[Conclusion] Our data reveals that the cloned CMVE-PEG3 and PEG-3 promoter are tumor-specific,and the CMVE-PEG3 promoter has higher transcriptional activities in prostate cancer DU145 cells,and may serve as a useful tool for transcriptional targeting gene therapy of human prostate cancer.
出处
《肿瘤学杂志》
CAS
2013年第7期548-552,共5页
Journal of Chinese Oncology
基金
国家自然科学基金资助项目(30800275)
