【作者】 张瑜；

【导师】 周建华；

【作者基本信息】 华中科技大学 ， 儿科肾脏， 2010， 博士

【摘要】 第一部分儿童HBV-MN肾组织细胞凋亡的研究目的观察儿童乙型肝炎病毒相关性膜性肾病(hepatitis B virus-associated membranous nephropathy, HBV-MN)肾组织的细胞凋亡情况,以探讨其在HBV-MN发病中的意义。方法采用免疫组织化学方法检测19例HBV-MN及5例正常肾组织caspase-3的表达,取细胞核呈棕黄色者为活性caspase-3阳性细胞,计数平均单个肾小球内阳性细胞比例及平均单位面积内肾小管-间质区阳性细胞个数,分别计为肾小球凋亡指数和肾小管-间质区凋亡指数,并分析其与肾组织病理改变、尿蛋白、及临床免疫学指标等因素的关系。同时对其中15例患儿行TUNEL检测,取细胞核呈棕黄色者为TUNEL阳性细胞,以前述方法计算肾小球及肾小管-间质区的TUNEL指数,并分析其与凋亡指数(由活性caspase-3所得)的相关性。采用免疫组织化学方法检测14例HBV-MN及3例正常肾组织HBx的表达。结果①正常肾组织,caspase-3仅见于少数肾小管上皮细胞胞浆,未见肾小球内表达；在HBV-MN患儿肾组织,均存在caspase-3的肾小球内表达,并且大多见于足细胞胞核区,而在肾小管-间质区caspase-3不仅见于肾小管上皮细胞胞浆,尚可见于胞核区,且表达的细胞数明显多于对照组；以TUNEL分析对活性caspase-3染色进行验证,发现肾小球和肾小管-间质区的凋亡指数与TUNEL指数均显著相关(r=0.812, P=0.0496 &r=0.958, P=0.0026);②HBV-MN患儿肾组织活性caspase-3的表达与肾脏病变病理分期显著相关,Ⅲ-Ⅳ期患儿的肾小球凋亡指数及肾小管-间质区凋亡指数均明显高于Ⅰ-Ⅱ期(P=0.015,0.049)；但肾小球凋亡指数与HBsAg的沉积强度无显著相关性；③HBV-MN患儿肾小球内活性Caspase-3的表达与临床蛋白尿水平相关,24h尿蛋白定量大于50mg/kg患儿的肾小球凋亡指数明显高于小于50mg/kg者(P=0.034),但与血清补体C3水平无显著相关性；④HBV-MN患儿肾小管-间质区凋亡指数与蛋白尿水平显著相关,24h尿蛋白定量大于50mg/kg患儿的阳性细胞数明显高于小于50mg/kg者(P=0.0046)；且与尿RBP水平相关,尿RBP升高组的活性caspase-3阳性细胞数显著高于尿RBP正常组(P=0.0012)。⑤71%HBV-GN患儿的肾小球内存在不同程度的HBx表达,其主要分布于肾小球毛细血管襻及足细胞胞浆。结论HBV-MN患儿肾小球及肾小管-间质区均存在依赖caspase-3的肾脏固有细胞凋亡,活性caspase-3的表达与肾脏病变严重程度、临床蛋白尿水平及尿RBP水平相关。肾脏固有细胞凋亡可能在HBV-MN的病理过程中发挥重要作用,而HBx蛋白可能与足细胞凋亡及进而的缺失密切相关。第二部分携带HBx基因重组腺病毒载体制备及其对足细胞凋亡的影响目的临床病理研究部分提示凋亡是HBV-MN患儿肾小球足细胞缺失的主要原因之一。而HBV-GN通常发生于HBV慢性感染患者,在此病理过程中HBV-DNA可与宿主DNA随机整合并发生基因重排,编码HBx等反式激活因子,进而诱导宿主细胞增殖或凋亡。因此,本部分研究旨在通过复制缺陷型腺病毒载体将HBx基因导入足细胞株,研究HBx对足细胞凋亡的直接效应。方法由质粒PCI-neo-HBx中扩增目的基因HBx,插入至pShuttle-CMV(-)穿梭质粒中,而后再与pAdxsi质粒进行酶切连接为重组腺病毒质粒pAd-HBx,经酶切及测序鉴定正确后,以PacⅠ酶切线性化转染293细胞进行包装扩增得到Ad. HBx。以CsCl密度梯度离心法纯化病毒,TCID50法检测病毒滴度。以瑞氏-吉姆萨染色观察细胞形态。Annexin V-PI双染方法检测足细胞凋亡。结果①重组腺病毒载体经限制性内切酶酶切、电泳和基因测序鉴定,证实Ad. HBx构建成功；②细胞形态学观察显示,腺病毒感染后24h,Ad. HBx组细胞的核分裂相明显增多甚至出现病理性核分裂相,巨核、双核、多核细胞的比例也较Ad组明显增加,但两组间的细胞凋亡率无明显差异；腺病毒感染后48h,Ad. HBx组细胞除上述有丝分裂障碍的病变更加明显外,胞体缩小、胞核浓染固缩的凋亡细胞比例较Ad组明显增多；③Annexin V-PI双染法显示,于腺病毒感染后24h,空白对照组、Ad组和Ad. HBx组三组间的凋亡细胞比例无明显差异；而于感染后48h,Ad. HBx组细胞的凋亡率较空白对照组和Ad组均明显升高(P<0.05)。结论成功构建了Ad. HBx为进一步研究HBx基因在乙型肝炎病毒相关性肾炎发病中的作用奠定了基础。外源性HBx表达可直接诱导足细胞凋亡。HBx可能是致HBV-GN患儿足细胞缺失的主要病因之一,在HBV-GN的发病中发挥着重要作用。第三部分外源性HBx表达抑制小鼠足细胞增殖目的乙型肝炎病毒相关性肾炎(hepatitis B virus-associated nephritis, HBV-GN)是一种以足细胞病变为特征的疾病。HBV-GN患儿肾活检组织中足细胞数目均存在不同程度减少,而增殖受限是足细胞缺失的主要机制之一。本研究通过构建复制缺陷型腺病毒载体将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MTT检测和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cyclin/DNA双参数流式细胞术和Western-blot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 blot显示,足细胞感染Ad.HBx(MOI=100)后第3、5天均可表达分子量为17kD的HBx蛋白,表明HBx可在足细胞株稳定表达。细胞形态学观察显示腺病毒感染后第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。MTT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；同时,CFSE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(增殖指数11.15)已明显低于空白组和Ad组(增殖指数15.4和13.3),且随时间推移进一步减慢(增殖指数32.5vs.61.65,53.96),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cyclin B1、p21的表达上调及cyclin A的下调。结论本部分研究显示,乙肝病毒基因编码的HBx蛋白可使cyclin B1降解受阻,同时下调cyclin A和上调细胞周期负调蛋白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HBV-GN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。更多还原

【Abstract】 PartⅠ. Apoptosis of resident renal cells in hepatitis B virus-associated membranous nephropathyObjective:To study apoptosis in renal tissues of children with hepatitis B virus-associated membranous nephropathy (HBV-MN) by using activated caspase-3 as a marker and explore the significance of renal inherent cells apoptosis in the pathogenesis of HBV-MN.Methods:Caspase-3 protein was specifically detected by immunohistochemical staining in 19 children with biopsy-proven HBV-MN and 5 normal patients suffering from nephrolithiasis or kidney trauma (control group).Brownish yellow nuclei were regarded as a marker of positive cells containing activated caspase-3 protein. In glomeruli and tubulointerstitial area, the positive cells were quantitated as glomerular and tubulointersititial apoptotic index respectively. And then both apoptotic indexes were correlated with clinical, serological and pathologic data. To confirm results obtained by activated caspase-3 detection, we used the TUNEL method to recognize apoptosis cells with brownish yellow nuclei in 15 children with HBV-MN.We took same manners to count TUNEL-positive cells and determine TUNEL index in glomeruli and tubulointerstitium, and analyzed the relationship between apoptotic index and TUNEL index. HBx protein was specifically detected by immunohistochemical staining in 14 children with HBV-MN.Results:①Caspase-3 protein expression was found in both cytoplasma and nuclei of glomerular cells and tubulointerstitial cells in HBV-MN,but only in cytoplasma of a few tubulointerstitial cells in controls.In glomeruli and tubulointerstitium, there was good correlation between apoptosis index assessed by activated caspase-3 staining and TUNEL index (r=0.812, P=0.0496 & r=0.958, P=0.0026).②The glomerular and tubulointerstitial apoptosis index were significantly correlated with pathologic severity of HBV-MN.The quantitative expression of activated caspase-3 was significantly higher in patients at phaseⅢorⅣthan those at phaseⅠorⅡ,but was not significantly different between patients with stronger HBsAg deposition (≥++) and those with weak deposition (<++).③Activated caspase-3 positive cells were much more in patients with massive proteinuria (≥50mg/kg) than those with moderate proteinuria (<50mg/kg, P=0.0335).But it wasn’t correlated with serum levels of C3.④Tubulointerstitial apoptosis index was significantly correlated with daily urine protein excretion and urine RBP levels.⑤HBx protein was detected within the glomerulus in 10/14 (71%)children with HBV-GN. The expression of HBx protein was localized within cytoplasm of podocytes and along the glomerular basement membrane in a fine linear pattern. Conclusion:All children with HBV-MN showed caspase-3-dependent apoptosis of renal intrinsic cells in glomeruli and tubulointerstitium. Furthermore, the expression level of activated caspase-3 was significantly correlated with renal pathologic severity, proteinuria and uric RBP level. Apoptosis of renal intrinsic cell may play an important role in the pathogenesis of HBV-MN in children. HBx may be an important cause of podocyte apoptosis in HBV-MN.PartⅡ.Construction of recombinant adenovirus containing HBx gene and its effect on podocyte apoptosisObjective Apoptosis has been regarded as an important mechanism of podocyte loss in HBV-associated membranous nephropathy. Since HBx has been demonstrated to be the major HBV protein causing host cell responses like proliferation or apoptosis, we hypothesize that HBx might be involved in the podocytopathy in HBV-MN.Therefore, in present study, hepatitis B virus X protein (HBx) was expressed in cultured mouse podocytes via adenoviral infection to determine its effect on podocyte apoptosis. Methods HBx cDNA was obtained from the plasmid PCI-neo-HBx by enzyme digestion, and inserted into shuttle plasmid pShuttle-CMV(-) to generate a recombinant plasmid pShuttle-HBx.Then the HBx gene was transferred from pShuttle-HBx to Adxsi viral DNA to form a recombinant adenoviral plasmid pAd-HBx by means of an in vitro ligation reaction. Finally, after identification, the pAd-HBx was packaged into infectious adenoviral particles Ad.HBx by transfecting human embryonic kidney (HEK) 293 cells. Cell morphologic changes were investigated by staining with Wright-Giemsa. HBx-induced podocyte apoptosis was demonstrated by flow cytometry. Results The recombinant adenovirus Ad.HBx was generated successfully by an in vitro ligation reaction, which was confirmed by restriction enzyme digestion and DNA sequencing. The HBx-expressing podocytes showed increased percentage of meganuclear and polynuclear cells at 24h, and significant apoptosis at 48h compared with control.In HBx-transfected group, the percentage of apoptotic podocytes (indicated by annexin V-posotive cells) increased 1.2-fold over control at 48h. Conclusions The successful construction of the recombinant adenovirus containing HBx gene has laid a foundation for further study of the involvement of HBx in the podocytopathy in HBV-GN. HBx can directly induce podocyte apoptosis, which may play an important role in the pathogenesis of HBV-GN.PartⅢ. Hepatitis B virus X protein reduces podocyte proliferation in vitroBackground Hepatitis B virus-associated nephritis (HBV-GN), characterized by podocyte injury, is a common secondary glomerular disease in China. Our previous studies have shown that podocyte number was significantly decreased in children with HBV-associated membranous glomerulopathy. Lack of proliferation has been regarded as one of the critical mechanisms contributing to injury-induced podocyte loss. Therefore, in current study, hepatitis B virus X protein (HBx) was expressed in cultured mouse podocytes via adenoviral infection to determine its effect on cell proliferation and its regulation at the level of cell cycle.Methods HBx gene was inserted into an adenovirus-based vector and transfected into mouse podocytes. Cell morphologic changes were investigated by staining with Wright-Giemsa. Cell growth was measured by MTT-assay and CFSE proliferation assay. Cell cycle phase was demonstrated by flow cytometry, and the expression of specific cell cycle regulatory proteins was examined by western blot analysis or flow cytometric bivariate 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 5 days post-infection, HBx-expressing podocytes presented with the characteristic of mitotic catastrophe such as binuclei,multinuclei,and polymorphonuclei accumulation. MTT assay showed that the growth curve of Ad-infected podocytes was basically consisten with controls, but both were significantly higher than the growth curve of Ad. HBx-infected podocytes after day 4 post-infection. Furthermore, CFSE-based proliferation assay also showed that at 3 days post-infection, the proliferation rate of HBx-expressing podocytes was significantly slower than Ad-infected podocytes and controls (proliferation index: 11.15 vs.15.4,13.3),and this trend became much obvious with the time (proliferation index: 32.5 vs.61.65,53.96).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 Our study shows that exogenous expression of HBx decreases the growth of podocytes. This effect is mediated through the G2/M phase arrest associated with an increase in the level of cyclin B1 and CDK-inhibitor p21 accompany with a decrease in the expression of cyclin A. These events may partially explain reduced podocyte number in HBV-GN.更多还原