| 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. |
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