树鼩慢性感染乙肝病毒过程中肝组织病理学观察及枯否细胞变化意义的探讨

  本文关键词：树鼩慢性感染乙肝病毒过程中肝组织病理学观察及枯否细胞变化意义的探讨，由笔耕文化传播整理发布。

        目的乙型肝炎病毒(Hepatitis B virus,HBV)感染是全球面临的重大公共卫生问题,是引起急慢性肝炎、肝硬化及肝癌的主要病原。建立一种简便、有效、稳定的HBV感染动物模型对于探索其感染机制、寻找有效的防治方法、研制抗HBV药物都具有重要的意义。本课题组前期研究证实,新生期接种HBV的树鼩能够长期感染HBV,并且HBV能够在树鼩体内稳定复制和长期存在。本研究拟在课题组以往研究的基础上,一方面继续观察慢性感染HBV的树鼩肝组织的病理组织学改变进展、分析和比较其与人类慢性感染HBV后的病理变化过程的异同,另一方面,为寻找树鼩HBV感染率的影响因素,本课题还对慢性感染HBV的树鼩肝组织的枯否细胞数量、功能及其可能的调节因子进行检测,以探索枯否细胞在树鼩感染HBV慢性化过程中的意义,为进一步优化树鼩模型提供线索。方法动物分为3组：A组,6只,为前期实验已确定慢性感染HBV(接种后1-6年)的树鼩；B组,3只,为前期实验疑似慢性感染HBV的树鼩(接种后3-4年)；C组,4只,为未接种HBV的正常对照树鼩。全部动物定期抽血和进行肝活检手术,采集的血清和肝组织标本分别作以下两部分研究。第一部分,分析慢性感染HBV的树鼩肝组织病理学变化,主要内容有：1.检测HBV感染指标,包括应用ELISA/TRFIA方法定性/定量检测HBV血清免疫学标志(两对半)、应用FQ-PCR定量检测血清和肝组织的HBVDNA水平、应用免疫组织化学方法检测肝组织中的HBsAg和HBcAg阳性肝细胞。2.观察肝组织病变,包括对组织切片应用HE染色,结合网状纤维、Masson等特殊染色及透射电镜等方法,观察肝脏病理组织学及细胞超微结构的改变,评价病变级别；同时,通过Ki67、P53和Cyclin D1免疫组化染色,评价肝细胞增殖水平。第二部分,分析慢性感染HBV的树鼩肝内枯否细胞的功能和数量变化,并分析其可能的影响因子。主要内容有：1.检测树鼩肝组织中枯否细胞数量变化,即应用流式细胞术检测从树鼩肝活检组织分离的肝非实质细胞中CD163+细胞的比例,以及用免疫组化染色方法原位检测树鼩肝组织中的枯否细胞数量。2.检测树鼩肝组织中枯否细胞的功能,即对手术切取的树鼩肝组织进行枯否细胞的分离、鉴定和原代培养,然后检测枯否细胞的迁移功能、吞噬功能及合成促炎症介质TNF-a的功能。其中,迁移功能的检测为以原代培养的枯否细胞开展细胞迁移实验,以及用免疫荧光染色法检测枯否细胞内的与迁移能力有关的细胞骨架成分微丝蛋白及微管蛋白的表达水平；吞噬功能的检测为应用溶酶体荧光探针检测原代培养的枯否细胞内溶酶体数量,以及用免疫组化方法原位检测肝组织中溶菌酶的表达情况；合成促炎症介质TNF-a功能的检测为应用VVestern blot及实时荧光定量RT-PCR方法,分别检测肝组织TNF-a蛋白及原代培养的枯否细胞TNF-a mRNA表达水平。3.检测树鼩肝组织中可能影响枯否细胞功能的因子,即应用实时荧光定量RT-PCR方法,检测原代培养的枯否细胞TLR-2及TLR-4等基因mRNA表达水平。结果第一部分,1.A组树鼩呈现HBV持续感染状态,6只动物于最后一次检测(接种HBV后1-6年)仍全部显示血清HBsAg和HBV DNA阳性、肝组织中有HBsAg阳性肝细胞；B组仅1只动物为血清HBsAg马阳性,其余指标阴性；C组全部动物的HBV感染标志均为阴性。2.A组各动物肝组织均有不同程度的慢性肝炎改变,表现为散在或弥漫分布的肝细胞水肿、脂肪变性、嗜酸性变,以及汇管区炎症——汇管区出现较明显的以淋巴细胞为主的炎细胞浸润,伴随小胆管增生；其中1只感染时间最长的动物(A1,接种HBV后6年)还出现小叶内多处坏死灶融合甚至桥接坏死及纤维化、大细胞性不典型增生等组织学改变。A组树鼩的肝活检组织学评分均值为5.33±3.93,显著高于B组(1分,P=-0.018)和C组(0分,P=0.008)；秩相关分析结果表明,组织学评分与动物HBV感染时长及其血清HBV DNA拷贝数呈显著的正相关关系(与感染时长的关系为r=0.808、P=0.000,与血清HBVDNA拷贝数的关系为r=0.494、P=0.014)。电镜下,A组动物的肝细胞超微结构变化表现为部分肝细胞肿大、表面微绒毛肿胀、溶酶体数量增多、胞质中糖原颗粒多少不均、线粒体肿胀变大、内质网扩张或形成不规则囊泡。免疫组化检测肝组织中的细胞增殖因子结果显示,A组动物的Ki67、P53和Cyclin D1表达水平均显著高于B组(分别为P=0.043、P=0.039和P=0.016)和C组(分别为P=0.050、P=0.021和P=0.007)；秩相关分析结果表明,肝组织的Ki67、P53及Cyclin D1的表达水平与肝组织学评分、血清HBV DNA考贝数及肝组织HBV DNA拷贝数均有显著的正相关关系(与组织学评分的关系,分别为r=0.829和P=0.000、r=0.815和P=0.001、r=0.913和P=0.000；与血清HBV DNA拷贝数的关系,分别为r=0.868和P=0.000、r=0.919和P=0.000、r=0.874和P=0.000；与肝组织HBV DNA拷贝数的关系,分别为r=0.744和P=0.004、r=0.846和P=0.000、r=0.876和P=0.000)。第二部分,树鼩肝组织分离枯否细胞的产量约为1.2±0.2x106个细胞/g肝脏,细胞活力为90%,纯度为85%。对原代培养的枯否细胞以及对肝组织中的枯否细胞原位检测结果显示：1.A组树鼩肝内枯否细胞数量增加——经流式细胞术测定,A、B、C三组CD163+细胞占肝非实质细胞的比例分别为89.80±0.36%、77.92±1.22%及77.97±1.13%,A组显著高于B组(P=0.034)和C组(P=0.021)；免疫组化检测结果显示,A、B、C三组肝组织内CD163阳性细胞计数分别为39.92±6.61、24.73±3.85和21.78±2.31个/高倍视野,A组显著高于B组(P=0.028)和C组(P=0.010)；秩相关分析结果显示,枯否细胞的数量与动物感染HBV时长及血清HBV DNA拷贝数存在正相关关系(与感染时长的关系为r=0.737、P=0.000,与血清HBV DNA拷贝数的关系为r=0.497、P=0.013)。2.A组树鼩枯否细胞功能下降——经细胞迁移实验检测,A、B、C三组的迁移细胞数均数分别为6.50±0.93、16.13±0.70和16.25±0.87,A组显著低于B组(P=0.034)和C组(P=0.021)；A组枯否细胞的微丝蛋白及微管蛋白的荧光强度、细胞溶酶体荧光强度、溶菌酶免疫组化阳性表达的细胞计均低于B组和C组(微丝蛋白,P=0.034和P=0.021；微管蛋白,P=0.034和P=0.021；溶酶体荧光,P=0.034和P=0.021；溶菌酶,P=0.020和P=0.011)；A组枯否细胞TNF-a mRNA的表达水平低于B组和C组(P=0.034和P=0.021),肝组织的TNF-a蛋白质表达水平也支持上述结果。秩相关分析结果显示,溶菌酶阳性细胞计数与动物感染HBV时长及其血清HBV DNA拷贝数均存在显著的负相关关系(与感染时长的关系为r=-0.890、P=0.000；与血清HBVDNA拷贝数的关系为r=-0.601、P=0.002),而TNF-a mRNA表达量与动物肝组织的HBV DNA拷贝数也呈负相关关系(r=-0.622、P=0.041)。3.检测可能影响枯否细胞功能的因子的结果显示,A组TLR-2mRNA及TLR-4mRNA表达水平均低于B组(P=0.034及P=0.021)和C组(P=0.034及P=0.021)。秩相关分析结果显示,TLR-2mRNA及TLR-4mRNA表达水平均与动物的肝组织HBV DNA拷贝数呈负相关关系(分别为r=-0.622、P=0.041和r=-0.673、P=0.023)；而该二基因的mRNA表达水平与枯否细胞的其它指标则均呈正相关关系,如与枯否细胞迁移数的关系(分别为r=0.809、P=0.003和r=0.845、P=0.001)、与溶酶体密度的关系(分别为r=0.745、P=0.008和r=0.609、P=0.047),以及与TNF-a mRNA表达水平的关系(分别为r=0.782、P=0.006和r=0.739、P=0.010)结论1..慢性感染HBV的树鼩在血清病毒学指标、组织病理学改变、超微结构特点及病程发展等方面与人类慢性感染HBV后的改变有诸多相似之处,此为其他同类动物模型所不具备的特点,是动物感染HBV研究领域的首次报道,提示树鼩感染HBV模型更适用于人类感染HBV相关的基础和临床研究。2.慢性感染HBV的树鼩肝组织损伤、肝细胞增殖指数分别与其感染HBV病程的长短、血清HBV DNA拷贝数呈显著的正相关关系,提示HBV在宿主体内的持续感染及复制可促进肝组织慢性病变的发展；对这些慢性感染HBV的树鼩继续进行观察,将有可能证明HBV是肝癌的独立诱发因素之一。3.慢性感染HBV的树鼩肝脏枯否细胞数量增加,但该细胞的迁移功能、吞噬功能及合成促炎症介质TNF-a等功能均下降,后者可能由该细胞表达TLR-2mRNA和TLR-4mRNA的水平下降所致；树鼩感染HBV的病程与其肝内枯否细胞的数量及功能变化显著相关。此系列结果提示,枯否细胞在宿主感染HBV的慢性化过程中可能起一定的调节作用。

    ObjectiveHepatitis B virus (HBV) infection is a major public health problem in the world. It is the major cause of acute and chronic hepatitis, cirrhosis and liver cancer. A simple, effective and stable HBV-infection animal model is impotent for exploring the infection mechanism, finding the effective prevention and treatment, and developing anti-HBV drugs. Our preliminary studies confirmed that neonatal tree shrews inoculated with HBV can become chronically infected with HBV, and HBV can replicate and exist in vivo stably for long. On the basis of previous studies, one of the purposes of this study is to examine the histopathological changes in the liver tissues of the tree shrews infected chronically with HBV, to compare the similarities with the pathological changes in the human liver of chronic HBV infection. On the other hand, in order to explore the significance of Kupffer cells in the process of HBV infection, this study is going to investigate the changes of Kupffer cells in the liver of tree shrew infected chronically with HBV, including the changes in number, function and the possible regulatory factors of Kupffer cells.MethodsAnimals were divided into three groups. Group A had6tree shrews that were identified as infected with HBV chronically, group B had3tree shrews that were suspected as infected with HBV chronically, while group C had4tree shrews that did not inoculated with HBV and were as normal controls. The serum and liver biopsy were collected regularly from all animals, the samples were then applied for the two-part study as following.Part one is to analyze the histopathological changes in the liver, including:(1) To detect the markers of HBV infection, such as to qualitatively/quantitatively detect the HBV serum immunological markers by the methods of ELISA and TRFIA, to quantitatively detect the level of HBV DNA in serum and liver tissue by FQ-PCR, and to immunohistochemically detect HBsAg and HBcAg in liver tissues.(2) To observation the histopathologic changes in liver tissues, by HE-stained tissues-slides, with some special staining such as reticular fibers stain and Masson’s stain, followed by evaluating the changes. Meanwhile, transmission electron microscopy was used to observation the ultrastructural changes in liver, and immunohistochemical stains for Ki67, P53and Cyclin D1were used to evaluate status of cell proliferation in liver.Part two is to analyze the changes in function and number of the intrahepatic Kupffer cells in tree shrews infected chronically with HBV, followed the isolation, identification and primary culture of Kupffer cells from liver tissue. The tests including:(1) To test the number of Kupffer cells, by flow cytometry to detect the proportion of CD163+cell within the non-parenchymal cells isolated from the tree shrew liver biopsy, as well as by immunohistochemical staining to detect in situ the proportion of Kupffer cells within the liver tissue of the tree shrew.(2) To detect the functions Kupffer cells such as migration, phagocytosis and synthesis of pro-inflammatory mediator TNF-a. The migration function were tested by cell migration assay on primary cultured Kupffer cells, as well as by immunofluorescence staining for cytoskeletal components microfilament and microglobulin of Kupffer cells. The phagocytosis function were tested by lysosomal fluorescent probe to detect the number of lysosomes on primary cultured Kupffer cells, as well as by immunohistochemical staining to detect the lysosomes on liver tissue samples. While the ability for synthesizing the pro-inflammatory mediators were detected by Western blot on liver tissue sample for TNF-a protein, and by real-time RT-PCR on primary cultured Kupffer cells for TNF-a mRNA, respectively.(3) To detect the factors that might affect the functions of Kupffer cell, by applying real-time RT-PCR on primary cultured Kupffer cells for the expression of TLR-2and TLR-4at mRNA levels.ResultsThe results of part one showed:(1) Tree shrews in group A presented persistent HBV infection, by showing positive HBsAg and HBV DNA in their serum as well as in liver tissues, which had lasted longer than3years after HBV vaccination. Only one animal in group B occasionally showed weakly positive serum HBsAg, while the remaining markers were negative. All animals in group C group were negative for the all markers of HBV infection.(2) Animals in group A showed different levels of chronic hepatitis change, such as scattered or diffuse distribution of liver cell edema, fatty and eosinophilic degeneration, as well as inflammation of the portal area which appeared obvious lymphocyte infiltration, accompanied by small bile duct hyperplasia. One animal (number A1, had been infected with HBV longer than six years) showed multiple necrosis which even fused to form bridging necrosis and fibrosis, as well as large cell dysplasia and other histological changes. The hepatic histological score of the animals in group A was5.33±3.93, which was higher than that in group B (1score, P=0.018) and group C (0score, P=0.008).Correlation analysis showed that the histological score was positively correlated with the duration of infection (r=0.808and P=0.000), and with the HBV DNA level in serum (r=0.494and P=0.014). Under electron microscope, ultrastructure changes in the liver cells of animal in group A were cell swell, microvilli swelling, lysosomal increase, uneven number of glycogen granules in the cytoplasm, mitochondrial swelling, endoplasmic reticulum expansion or the formation of irregular vesicles. Immunohistochemical detection for cell proliferation factor showed that the expression levels of Ki67, P53and cyclin D1of group A group were all higher than those in group B (P=0.043, P=0.039and P=0.016, respectively) and group C (P=0.050, P=0.021and P=0.007, respectively). Correlation analysis showed that Ki67, P53and cyclin D1expression levels in liver tissue were significantly and positively correlated with the liver histological scores (r=0.829and P=0.000, r=0.815and P=0.001, r=0.913and P=0.000, respectively), serum HBV DNA copy number (r=0.868and P=0.027, r=0.919and P=0.000, r=0.874and P=0.000, respectively), and the HBV DNA copy number in liver tissues (r=0.744and P=0.004, r=0.846and P=0.000, r=0.876, P=0.000, respectively).The results of part two showed that the harvest of Kupffer cell from tree shrew liver tissue were1.2±0.2×106cells/g liver, the cell viability was90%and the purity was85%. The results of tests on Kupffer cells in primary culture, as well as of the in the liver tissue showed:(1) The number of Kupffer cells increased in the tree shrews of group A. Measured by flow cytometry, CD163+cells within the non-parenchyma cells of group A, B, C were89.80±0.36%、77.92±1.22%and77.97±1.13%, respectively. The number of group A was higher significantly than that of group B (P=0.034) and group C (P=0.021). Immunohistochemistry test results showed that the CD163-positive cells in group A, B and C were39.92±6.61,24.73±3.85and21.78±2.31/high power field, respectively. The number of group A was higher than that of group B (P=0.028) and group C (P=0.010) too. Correlation analysis showed that the number of Kupffer cells was positively correlated with the duration of animals infected with HBV (r=0.737and P=0.000), as well as with the level of HBV DNA in serum (r=0.479and P=0.013).(2) Function of Kupffer cells reduced in the animals of group A. The average numbers of migrating cells in group A, B and C were6.50±0.93,16.13±0.70and16.25±0.87, respectively. The number in group A was lower than that in group B (P=0.034) and C group (P=0.021). Meanwhile, group A showed lower levels than group B and C in the fluorescence intensities of microfilament protein (P=0.034and P=0.021respectively), of microtubule protein (P=0.034and P=0.021respectively), of lysosomal (P=0.034and P=0.021respectively), and in the number of lysozyme-positive cells detected by immunohistochemistry (P=0.020and P=0.011respectively). Also, the TNF-a mRNA expression level of Kupffer cell in group A was lower than that in group B (P=0.034) and group C (P=0.021), while the TNF-a protein expression levels in liver tissue tested by Western blot showed the similar tendency. Correlation analysis showed that the number of lysozyme-positive cells was significantly and negatively correlated with the duration of animal infected with HBV (r=-0.892and P=0.000), the level of HBV DNA in serum (r=-0.601and P=0.002). Meanwhile, expression level of TNF-a mRNA animal was negatively correlated with the levels of HBV DNA in liver tissues (r=-0.622and P=0.041).(3) Result of the detection on the factors that might affect the function of Kupffer cell showed expression levels of TLR-2mRNA and TLR-4mRNA in group A were lower than those in group B (P=0.034and P=0.021, respectively) and C group (P=0.034and P=0.021, respectively). Correlation analysis showed that the expression levels of TLR-2mRNA and TLR-4mRNA were negatively related with the level of HBV DNA in liver tissues (r=-0.622and P=0.041, r=-0.673and P=0.023, respectively). However, the expression levels of TLR-2mRNA and TLR-4mRNA were positively related with some other factors, such as the number of migrated Kupffer cells (r=0.809and P=0.003, r=0.845and P=0.001, respectively), the density of lysosomes relationship (r=0.745and P=0.008, r=0.609and P=0.047, respectively), and the expression level of TNF-a mRNA (r=0.782and P=0.006, r=0.739and P=0.010, respectively).Conclusions1. Tree shrew with chronic infection of HBV is similar to human beings based on the serum virological indicators, histopathological changes, ultrastructural characteristics and progression of infection. These characters are unique in tree shrews than other animals. Therefore, tree shrews can be used as a research model of chronic HBV infection.2. In tree shrews with chronic HBV infection, liver histology score, liver cell proliferation index, infection time and serum HBV DNA copy number were positively correlated. These results suggested that the liver histological changes in tree shrews with chronic HBV infection correlated with persistent HBV replication and infection 3. Kupffer cells number increase in tree shrews with chronic HBV infection; however, the cell migration, phagocytosis function and TNF-α synthesis function declined. It suggested that the course of the tree shrews chronic HBV infection correlated with Kupffer cell volume and function changes4. Decreased expression of TLR-2and TLR-4mRNA was observed in Kupffer cells from the tree shrew with chronic HBV infection. It is possible reason that Kupffer cells showed a decrease of migration function, phagocytosis and TNF-a synthesis function.

树鼩慢性感染乙肝病毒过程中肝组织病理学观察及枯否细胞变化意义的探讨

中英文缩略词4-5摘要5-10ABSTRACT10-16前言17-34材料与方法34-62结果与讨论62-113    1、第一部分结果62-77    2、第一部分讨论77-85    3、第二部分结果85-105    4、第二部分讨论105-113结论113-114问题与展望114-115参考文献115-125综述：固有免疫在乙型肝炎病毒感染中的作用125-142    参考文献136-142致谢142-143攻读学位期间发表的学术论文143

  本文关键词：树鼩慢性感染乙肝病毒过程中肝组织病理学观察及枯否细胞变化意义的探讨，由笔耕文化传播整理发布。