【作者】 赵月；

【导师】 佘锐萍； 唐军；

【作者基本信息】 中国农业大学 ， 基础兽医学， 2014， 博士

【摘要】 乙型肝炎(HB)是由乙型肝炎病毒(HBV)引起的一种以肝脏损伤为主要特征的严重危害人类健康的传染病之一。由于HBV具有严格的宿主特异性,因而缺乏理想的细胞模型或动物模型,从而阻碍了HBV的研究。本研究采用人源HBV对沙鼠和C57BL/6小鼠进行感染试验,目的在于探讨人源HBV能否引起小鼠感染,并探讨小鼠作为研究HBV传播和致病机制的动物模型的可能性。本研究选取沙鼠和C57BL/6小鼠用作人源HBV阳性血清的人工感染试验动物,采用ELISA、PCR、Real-time PCR、免疫组织化学和Western blot等方法对人源HBV感染后沙鼠和C57BL/6小鼠的血清、粪便和肝脏等组织进行HBV相关病原和抗体的检测,来探讨HBV在沙鼠和C57BL/6小鼠体内的感染情况,并通过沙鼠和C57BL/6小鼠体内细胞凋亡和内质网应激相关蛋白的定位和表达,初步揭示HBV感染致肝细胞损伤的机制。研究结果如下：1、人源HBV感染沙鼠后1d,在肝脏和肾脏组织中可检测到HBV DNA;感染后2d和4w,只在肝脏中检测到HBV DNA。感染C57BL/6小鼠后1d,肝脏中可检测到HBV DNA。在血清、粪便、脑组织和唾液腺中,未能检测到HBV DNA。血清ELISA检测结果表明,在感染后1w内,HBsAg和HBeAg在小鼠血清中呈一过性出现；感染后1w,在沙鼠和C57BL/6小鼠的血清中均可检测到HBsAb和HBcAb,而且HBsAb能够在整个试验过程中持续性在血清中被检测到。肝脏组织中HBV相关抗原免疫组化染色结果表明沙鼠和C57小鼠感染人源HBV后肝脏中可检测到抗原阳性信号,且强度随感染时间的延长而增强。Western blot检测发现在感染后7d沙鼠肝脏中HBV抗原PreS1蛋白有明显的表达。2、沙鼠和C57BL/6小鼠感染人源HBV后均出现明显的组织病理学变化,呈现典型的病毒性肝炎的病理学变化。主要表现为肝窦扩张、淤血,肝细胞胞浆疏松、颗粒变性,汇管区淋巴细胞浸润和胆管增生等,病变程度随感染时间的延长而显严重。电镜观察发现,试验组小鼠肝细胞结构松散,内质网扩张,线粒体出现严重肿胀、空化。肾小管.上皮细胞胞质疏松,线粒体肿胀、嵴消失,内质网囊泡化,可见髓鞘样小体,胞浆内可见病毒包涵体,胞核内可见病毒样粒子。3、肝脏组织中增殖与修复相关蛋白免疫组化染色结果表明HBV感染后,沙鼠和C57BL/6小鼠肝脏组织中PCNA和HSP70蛋白在体内表达出现先升高后下降的趋势,攻毒后蛋白表达显著高于对照组(P<0.05),表明HBV感染后机体出现了相应的抗损伤机制来减少损害。4、细胞凋亡相关分子的免疫组化染色结果表明感染后沙鼠和C57BL/6小鼠体内Bax、Bcl-2、 Caspase-3和Fas/FasL的表达量均有不同程度的升高,显著高于对照组(P<0.05),表明HBV感染后细胞凋亡的线粒体途径被激活,细胞凋亡在HBV感染导致肝细胞损伤的机制中发挥了重要作用。5、内质网应激(ERS)相关分子免疫组化染色结果表明感染后沙鼠和C57BL/6小鼠肝脏组织中GRP78、CHOP、JNK及Caspase-12的表达均显著高于对照组(P<0.05)。Western blot结果也证实感染后肝脏ERS相关分子的表达量较对照组均有所升高。ERS相关蛋白表达量的升高表明HBV感染后启动了内质网应激反应,促进了肝细胞的凋亡。6、对沙鼠肝脏线粒体DNA不同区域基因序列扩增,观察、分析比较了人源HBV感染8w后,沙鼠肝脏线粒体DNA水平的变化,发现人源HBV感染后在沙鼠肝脏线粒体DNA的D-loop、 CytB和CytC三个区域均出现了一个碱基位点的改变,这些改变可能与线粒体结构或功能的变化相关。上述研究结果表明,人源HBV感染可引起沙鼠和C57BL/6小鼠肝脏组织发生明显的病理损伤和炎症反应,采用PCR、血清ELISA和肝脏免疫组织化学检测在感染鼠体内检到HBV DNA及其相关抗原的存在,HBV可在小鼠体内进行短期的复制表达,表明沙鼠和C57BL/6小鼠具有作为HBV感染动物模型的潜质。通过对HBV感染后沙鼠和C57BL/6小鼠肝脏的细胞损伤、细胞凋亡和内质网应激等相关因子表达量的变化初步观察研究发现,HBV感染后可通过激活线粒体途径和内质网应激途径促进肝细胞的凋亡,这可能是HBV感染引起肝细胞损伤的重要机制。更多还原

【Abstract】 Hepatitis B virus (HBV) infection, characterized by liver injury, caused public health problem worldwide. The studies on HBV have long been hampered by the strict host specificity and lack of appropriate cell models or animal models. To explore the possibility of animals being infected by human HBV, Mongolian gerbils and C57BL/6mice were inoculated with human HBV. A variety of approaches including ELISA, PCR, Real-time PCR, immunohistochemistry staining and western blot were used for detection of HBV related antigens and antibodies in serum, faces and liver samples. Proteins involved in apoptosis and endoplasmic reticulum stress (ERS) were analyzed to explore the mechanism of liver injury caused by HBV inoculation. The results are as follows:1、After inoculated with human HBV, the liver and kidney tissues of gerbils were detected to be HBV DNA positive1d post inoculation (dpi), however only the livers were HBV DNApositive at2dpi and4w dpi. In addition, HBV DNA was detected positive in the liver tissues of C57BL/6mice but only at1dpi. However, HBV DNA were fail to be positive in serum, faeces, brain and salivary glands.ELISA results demonstrated that HBsAg and HBeAg were transiently existed in the serum of gerbils and C57BL/6mice within1w after inoculation, while the HBsAb and HBcAb were detected till8w. Immunohistochemical analysis showed the persistent presence of HBV antigens in livers of gerbils and C57BL/6mice after inoculation.The signals of HBV related antigens were increased with the inoculation time. Western blot analysis revealed the presence of PreS1in the liver tissues at7dpi of both gerbils and C57BL/6mice after inoculation with HBV.2、Obvious histopathological changes resembled viral hepatitis were observed in gerbils and C57BL/6mice after inoculated with human HBV. With dilated sinus and congestion in liver, swollen hepatocytes with extensive cytoplasmic vascuolation were obviously observed. Besides, lymphocytes infiltration around portal area and biliary hyperplasia proliferation were also observed.The lesions were aggravated with the time after inoculation. The hepatic ultrastructure observed by TEM demonstrated loose cytoplasmic matrixes in hepatocytes with marked changes in endoplasmic reticulum and mitochondria. The mitochondria were dilated with dissolved cristae, some of which formed concentric lamellar bodies in cytoplasm. Besides, spherical viral particles morphologically similar to HBV particles were observed in the nucleus of hepatocyte and the cytoplasm of epitheliums of renal tubules.3、Immunohistochemical staining results showed that the expression levels of PCNA and HSP70were increased at the initial stage and then slowly declined. The expression levels of the two proteins were significantly higher than that of the control group, suggesting that protective mechanisms were induced in response to the liver damage induced by HBV inoculation.4、The immunohistochemical staining results showed that the expressions of the apoptosis-related molecules Bax, Bcl-2, Caspase3and Fas/FasL were significantly higher in livers of HBV inoculated group than that of control (P<0.05), indicating the engagement of apoptotic pathways.5、The immunohistochemical staining results showed the expressions of the ERS associated GRP78and CHOP were significantly higher in livers of HBV inoculated group than that of control group (P<0.05). The expressions of JNK and Caspase-12in the inoculated group were also increased. Western blot analysis further confirmed the increased expressions of the ERS related proteins, suggesting that ERS-induced apoptosis in hepatocytes occurred after HBV inoculation.6、Mitochondria related genes were amplified and analyzed. One mutation in the D-loop of CytB and CytC mtDNA was revealed in the inoculated geibels, which might be associated with injuries of mitochondria structurally and functionally.In conclusion, the results showed that human HBV could induce hepatic pathological damage in gerbils and C57BL/6mice. ELISA and PCR analysis demonstrated the existence of HBV DNA and HBV related antigens and antibodies after inoculation. The expressions of apoptosis and ERS related proteins were increased after HBV inoculation, indicating apoptotic and ERS pathways were mobilized in response to HBV inoculation and liver injury.更多还原