摘要
目的探讨乙型肝炎病毒（HBV）X基因表达蛋白HBX对体外培养的人近端肾小管上皮细胞株（HK-2）细胞形态及转分化的影响。方法用分子克隆的方法构建pcDNA3．1-myc—HBX质粒，采用脂质体转染法瞬时转染HK．2细胞，Q-PCR及Western印迹法验证HBX在HK-2细胞中的表达。以未转染质粒和转染空载质粒pcDNA3．1-myc作为对照。用显微镜观察转染pcDNA3．1-myc—HBX质粒后HK-2细胞形态，用Western印迹及Q—PCR法检测细胞转分化标志蛋白α平滑肌肌动蛋白（α-SMA）、E钙黏蛋白（E—cadherin）的表达；ELISA检测细胞上清液中白细胞介素（IL）-1、IL-6和肿瘤坏死因子仅（TNF—α）的表达。结果转染I-IBX后的HK．2细胞中存在HBX的高表达，证实转染成功。转染pcDNA3．1．myc—HBX质粒的HK-2细胞数量明显下降，细胞形态不规则，细胞状态受损；转染pcDNA3．1-myc-HBX质粒的HK-2细胞可上调E．cadherin及α-SMA表达；细胞上清液中高表达IL-1、IL-6和TNF-α（P〈0．01）。结论HBX质粒转染HK-2细胞后可引起细胞数目和形态的变化，并促进肾小管上皮细胞发生上皮间质转分化，肾小管上皮细胞周围炎性微环境的改变可能是其发生上皮间质转分化的原因之一。
Objective To investigate the effects of hepatitis B virus X （HBX） gene on cell morphology and transdifferentiation of human proximal tubular epithelial cells. Methods The eukaryotic vector pcDNA3.1-myc-HBX containing HBX gene was transiently transfected into HK-2 cells by lipofectamine mediation. The expression of HBX was confirmed by Q-PCR and Western blotting. Untransfected HK-2 cells and those transfected with empty vector were used as control. The morphology of HK-2 cells was observed by microscopy, the expressions of differentiation marker proteins α- SMA and E-cadherin were detected by Western blotting and Q-PCR, and the contents of IL-1, IL-6 and TNF-α in the supernatant were detected by ELISA assay. Results HBX was successfully expressed in HK-2 cells after transfection. After transfection of HBX gene, the shape of HK-2 cells became irregular, HK-2 cells significantly expressed E-cadherin and a-SMA, and had high levels of IL-1, IL-6 and TNF-α in the cell supernatant （P〈0.01）. Conclusion Overexpression of HBX gene in renal tubular epithelial cells may damage cell morphology and promote the occurrence of epithelial- mesenchymal transdifferentiation, which may be related to the inflammatory microenvironment.
出处
《中华肾脏病杂志》
CAS
CSCD
北大核心
2012年第12期956-960,共5页
Chinese Journal of Nephrology
基金
基金项目：国家自然科学基金（81170672）
关键词
转染
炎症
肝炎病毒
乙型
肾小管上皮细胞
转分化
Transfection
Inflammation
Hepatitis B virus
Renal tubular epithelial cells
Transdifferentiation
