WT1基因启动子和增强子在不同细胞株中的转录活性研究  

Transcriptional Activity of WT1 Gene Promoter and Enhancer in Diverse Cell Lines

作　　者：胡绍燕[1] 陈子兴[1] 赵晔[1] 岑建农[1] 谷敏[1] 傅铮铮[1] 何军[1] 顾伟英[1] 

机构地区：[1]苏州大学附属第一医院,江苏省血液研究所,苏州215006

出　　处：《中国实验血液学杂志》2007年第5期1050-1055,共6页Journal of Experimental Hematology

基　　金：国家自然科学基金资助项目;编号39770306

摘　　要：本研究探讨WT1基因启动子和增强子在不同细胞株中的转录活性,为开展基于WT1基因调控的白血病基因治疗奠定基础。以EGFP做报告基因,构建含有WT1基因启动子和增强子的重组表达载体;利用脂质体和电穿孔技术将重组质粒转染13个细胞株,包括WT1基因高表达的白血病细胞株(K562、NB4、THP-1、SHI-1),WT1基因低表达的白血病细胞株(U937和Jurkat);非造血细胞株中WT1基因高表达的MCF-7、T47D、293株细胞和WT1基因低表达的ECV304、SMMC7721、HT-29、SHG44细胞株;利用流式细胞术检测转基因细胞稳定表达EGFP的平均荧光强度。以平均荧光强度代表启动子和(或)增强子的转录活性。结果表明:通过基因重组技术构建了含有WT1基因启动子的表达载体pEWP,以及含有WT1基因启动子和增强子的表达载体pEWPA。就启动子的转录活性而言,在非白血病细胞株中,ECV304细胞EGFP的平均荧光强度最高,是空载体(pEGFP-1)的16.54±2.45倍,明显高于白血病细胞株(p<0.05);MCF-7和SHG44细胞次之,分别为9.46±1.10和7.29±0.73倍,HT-29细胞表达最低,仅为0.99±0.02倍。在人类血病细胞株中,K562细胞EGFP的平均荧光强度最高,是空载体pEGFP-1的2.93±0.27倍,明显高于Jurkat和SHI-1细胞株(p<0.05)。后二者分别为0.74±0.03和0.84±0.09倍。pEWPA可以使HT-29、SHI-1和K562细胞中WT1基因启动子的转录活性增强,分别增强到4.81、3.06和1.01倍。结论:WT1基因启动子的转录活性与细胞内在WT1基因的表达水平不相关,增强子增强部分细胞株中WT1基因启动子的转录活性,但不具有造血组织特异性。The objective of study was to investigate tissue-specific transcriptional activity of WT1 （Wilms＇ tumor gene） promoter and enhancer in cell lines with diverse tissue origin for leukemic gene therapy depending on WT1 transcriptional regulation elements. WT1 promoter and enhancer were ligated into pEGFP-1 to construct a recombinant vectors with EGFP gene as a reporter. By using electroporation or lipofectamine, the resultant constructs were transfected into 13 cell lines including WTl-expressing hematopoietic cell lines （ K562, NB4, THP-1 and SHI-1 ）, WT1 -nonexpressing hematopoietic cell lines （U937 and Jurkat）, WTl-expressing nonhematopoietic cell lines （MCF-7, T47D and 293 ） and WT1-nonexpressing nonhematopoietic cell lines （ECV304, SMMC7721, HT-29 and SHG44）. The mean fluorescence intensity （MFI） of EGFP representing the transcriptional activities of promoter and/or enhancer was analyzed by using flow cytometry in the transfected cells which stably expressed EGFP. The results indcated that the vectors, pEWP containing WT1 promoter and pEWPA containing both WT1 enhancer and promoter, were constructed by recombinant DNA technique. Among nonhematopoietic cell lines, pEWP induced the highest EGFP expression in ECV304 （ 16.54 ± 2.45 times as high as pEGFP- 1 ）, mildly higher in MCF-7 and SHG44 （ 9.46 ± 1.10 and 7.29 ± 0.73 times of pEGFP- 1 level）, and lowest in HT-29 （0.99 20.02 times as much as pEGFP-1 ） respectively. Among hematopoietic cell lines, EGFP expression was highest in K562 cell line （2.93 ± 0.27 times of pEGFP-1 ）, which was statistically higher than those in Jurkat and SHI-1 cell lines （0.74 ± 0.03 and 0.84 ± 0.09 times of pEGFP-1 level） respectively, pEWPA, with WT1 enhancer inserted at All II site near SV40 polyA, increased basal transcription levels of the WT1 promoter in HT- 29, SHI-1 and K562 cells by 4.81, 3.06 and 1.01-fold respectively. It is concluded that the transcriptional activities of WT1 promoter in the recom-binant vector seem unrelat

关 键 词：WT1基因 WT1基因启动子 WT1基因增强子 平均荧光强度 白血病细胞株 造血组织特异性 

分 类 号：R733.7[医药卫生—肿瘤]