瞬时转染HBx基因到人正常胆管上皮细胞并观测其对hTERT mRNA的表达影响  

Transient transfection of HBV X gene into human normal biliary epithelial cells and its effects on expression of human telomerase reverse transcriptase mRNA

作　　者：屈振亮[1] 邹声泉[1] 王欣[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院普外科

出　　处：《中华肝胆外科杂志》2005年第6期411-413,共3页Chinese Journal of Hepatobiliary Surgery

基　　金：国家863攻关计划(2002AA214061)资助项目

摘　　要：目的通过研究HBx基因转染到人正常胆管上皮(HBECs)后对人端粒酶逆转录酶(hTERT)基因的转录调节作用,以阐明HBV感染在胆管癌发生中的作用机制。方法瞬时转染HBx基因到体外培养的HBECs,同时转染含增强型绿色荧光蛋白(EGFP)基因的克隆载体以判定转染率;RT-PCR分析转染后HBECs内hTERT mRNA的表达变化;并通过细胞免疫组化技术了解转染细胞内HBx蛋白的表达。结果经EGFP判定的转染率约为15%;未经转染或转染空载体的HBECs不表达hTERT mRNA,而转染HBx基因的HBECs可表达明显的hTERT mRNA;只有在转染了HBx基因的HBECs可检测到HBx蛋白表达。结论HBx基因转染能激活hTERT mRNA的转录表达,这种顺式调节作用可能是胆管上皮增殖、分化并恶化的主要机制。Objective To study the transcriptional regulation of human telomerase reverse transcriptase (hTERT) mRNA in human normal biliary epithelial cells (HBECs) after HBV X gene transfection and to elucidate the possible mechanism of HBV infection in the bile duct carcinogenesis. Methods HBV X gene was transiently transfected into HBECs and the cloning vector containing enhanced green fluorescent protein gene was co-cultured in order to determine the transfection efficiency. The expression of hTERT mRNA in HBECs was assayed by RT-PCR. The expression of HBV X protein in HBECs was detected by immunocytochemical staining. Results The transfection efficiency was about 15%. There was no expression of hTERT mRNA in HBECs when transfected with OPTI-MEM medium and blank vector. However, a dramatic expression of hTERT mRNA was observed in HBECs when transfected with HBV X expression vector. The expression of HBV X protein was only found in those transfected with HBV X expression vector. Conclusions HBV X gene transfection can activate the transcriptional expression of hTERT mRNA. The cis-activation of hTERT gene by HBX is the primary mechanism for proliferation, differentiation and tumorigenesis of biliary epithelia.

关 键 词：HTERT HBX基因 mRNA 瞬时转染 胆管上皮细胞 增强型绿色荧光蛋白 人端粒酶逆转录酶 观测 免疫组化技术 调节作用 基因转染 HBV感染 PCR分析 HBX蛋白 作用机制 体外培养 克隆载体 表达变化 EGFP 蛋白表达 转录表达 上皮增殖 

分 类 号：R575.6[医药卫生—消化系统]