中文摘要：

目的通过构建复制缺陷型腺病毒载体将乙型肝炎病毒（HBV）X基因（即HBx基因）导入足细胞株，探讨HBx蛋白对足细胞增殖的影响及其分子机制。方法采用pAdxsi系统构建携带HBx基因的复制缺陷型腺病毒载体。以瑞氏-吉姆萨染色观察细胞形态。以MTF检测和羧基荧光素二乙酸盐琥珀酰亚胺酯（CFDA—SE）荧光染料示踪细胞增殖的方法，对细胞增殖进行定量评估。采用流式细胞仪检测细胞周期分布。采用cyelin／DNA双参数流式细胞术和Western印迹检测细胞周期调控蛋白的表达。结果PCR和基因测序鉴定证实，Ad，HBx构建成功。Western印迹显示，足细胞感染Ad．HBx（感染复数MOI=100）后第3、5天均可表达相对分子质量为17000的HBx蛋白，表明HBx可在足细胞株稳定表达。细胞形态学观察最示腺病毒感染后第5天Ad．HBx组细胞呈现明显的有丝分裂障碍特征，双核、多核和多形核细胞比例明显增加。MTF检测结果显示空白组和Ad组足细胞的生长曲线基本吻合，而Ad．HBx组细胞的生长曲线自第4天起较前两组偏低，至第5天时该差异具有统计学意义（P〈0．01）；同时，CFDA—SE细胞增殖示踪实验也证实，于腺病毒感染后第3天Ad．HBx组细胞的增殖速度已明显低于空白组和Ad组（增殖指数11．2比15．4、13．3），且随时间推移进一步减慢（增殖指数32．5比61．6、54．0），表明HBx可显著抑制足细胞增殖。细胞周期分析和细胞周期调控蛋白检测的结果显示，HBx可诱导足细胞周期G2／M阻滞，同时伴有cyclinB1、p21的表达上调及cyclinA的下调。结论乙肝病毒基因编码的HBx蛋白可使eyclinB1降解受阻，同时下调cyolinA和上调细胞周期负凋蛋白p21的表达，导致细胞周期阻滞于G2／M期，从而抑制足细胞增殖。这可能是乙型肝炎病毒相关性肾炎患者肾小球足细胞缺失，肾脏病变慢性进展的重要机制之一。

英文摘要：

Objective To investigte the effect of hepatitis B virus X protein （HBx） expressed in cultured mouse podocytes via adenoviral infection on cell proliferation and to elucidate its possible mechanism. Methods HBx gene was inserted into an adenovirus-hased vector and transfected into mouse podocytes. Cell morphologic changes were investigated by staining with Wright-Giemsa. Cell growth was measured by MTI＇-assay and CFDA-SE proliferation assay. Cell cycle phase was demonstrated by flow cytometry, and the expression of specific cell cycle regulat（uy proteins was examined hy Western blot analysis or flow cytometric hivariate analysis. Results The recombinant adenovirus Ad.HBx was generated successfully, which was confirmed by polymerase chain reaction and DNA sequencing. The expression of HBx protein in the Ad. HBx- infected podocytes was confirmed by Western blot. At day 5 post-infection, HBx-expressing podocytes presented the characteristics of mitotic catastrophe such as binuclei, muhinnclei, and polymorphonuclei accumulation. MTT assay showed that the growth curve of Ad-infected podocytes was basically consistent with control, but both were significantly higher than that of Ad.HBxinfected podocytes after day 4 post-infection （P〈0.01）. Furthermore, CFDA-SE-based proliferation assay also revealed that at day 3 post-infection, the proliferation rate of HBx-expressing podocytes was significantly slower than that of Ad-infected podocytes and controls （proliferation index： 11.2 vs 15.4, 13.3）, and this trend became much obvious with the time （proliferation index： 32.5 vs 61.6, 54.0）. FACS analysis showed that with the HBx-induced G2/M phase arrest, the levels of cyclin B1 and CDK-inhibitor p21 were significantly increased in HBx-expressing podocytes, but the expression of cyclin A was slightly decreased. Conclusions Exogenous expression of HBx inhibits the growth of podocytes. This effect is mediated through the G2/M phase arrest associated with an increase of cyclin BI and CDK-inhibitor p21 and