腺病毒5型E1A基因转染前后乳腺癌细胞相关指标水平的变化  

Eukaryotic cell expression of E1A gene and study on effects on human breast cancer

作　　者：杨新宇[1] 宫路路[1] 刘志毅[2] 房学东[1] 

机构地区：[1]吉林大学第二医院普通外科,吉林长春130033 [2]吉林大学第三医院泌尿外科

出　　处：《中国实验诊断学》2007年第7期860-863,共4页Chinese Journal of Laboratory Diagnosis

摘　　要：目的本研究旨在探讨E1A基因对乳腺癌细胞凋亡的影响,为乳腺癌E1A基因治疗的可行性提供理论和实验依据。方法①构建E1A基因真核表达质粒pEGFP-E1A:通过酶切反应获得E1A基因,琼脂糖凝胶回收后,连接入pEGFP-C1载体EcoRⅠ和BamHⅠ位点中。②E1A基因转染乳腺癌细胞系:脂质体介导将E1A基因转入乳腺癌细胞系SK-BR-3细胞株,经筛选获得稳定表达E1A基因的转染试验组细胞。③RT-PCR检测E1A基因的mRNA表达、west-ern-blot检测E1A蛋白表达。流式细胞仪观察细胞凋亡率、免疫组化法检测HER-2/neu基因的表达水平,对比各组间的差异。结果与对照组、空载体组相比,转染E1A基因后,ElA RNA和蛋白表达明显增高。SK-BR-3肿瘤细胞HER-2/neu的表达明显降低,凋亡率增高。结论本实验成功构建并稳定表达了E1A基因质粒,明显抑制乳腺癌细胞的生长,说明E1A基因在乳腺癌基因治疗中有重要意义,为其临床应用提供了理论和实验依据。Objectlve Aim To research the effect of Ad5E1A gene on apoptosis of the hurman breast cancer. Methods ①E1A gene recombinant expression vector plasmid was constructed by subcloning a 1.77kb E1A gene into a mammalian expression vector pEGFP-C1 at the sites of EcoR Ⅰ and BamH Ⅰ . The recombinant was cleaved with appropriate endoneuclear and sequenced. ②Integratuion and expression of E1A gene in Human breast cancer cell line （SK-BR-3） by LipofectinTM Reagent. Obtain positive cell clones （SK-BR-3-E1A）.③The expressing change of HER-2/neu gene was investigated.4 Effects of adenovirus type 5 E/A gene to carcinoma ceils in vivo . Results The results showed that the orientation of the insert was found to be correct, while no rearrangement was found. E1A gene mRNA RT-PCR assay, westeru-blot assay showed that E1A gene Ires been integrated into ceils and stably expressed. Immunoeytoehemistry analysis showed that the products of HER-2/neu gene have decreased in E1A-expressing cells. Conclusion The above results showed that E1A gene possesses important effects on gene therapy of human breast carcinoma. Our study provided data for gene therapy of tumor and possible of using E1A gene to improve tumor treatment.

关 键 词：E1A基因 乳腺癌 基因转染 

分 类 号：R737.9[医药卫生—肿瘤]