Relationship Between Human Cytomegalovirus And CD4~+T Cell's Cytokines In Biliary Atresia

Objectives1. To detect the infection rate of several hepatotropic DNA viruses in the liver tissue of biliary atresia patients, and explore the associated viruses with the etiology of biliary atresia, then observe it's impact on the morphology of biliary epithelial cells in culture;2. To explore the correlation between human cytomegalovirus and CD4+T cell's cytokins in biliary atresia.Methods1. clinical samplesFresh liver tissue samples were taken from 85 cases(50 male and 35 female) of biliary atresia patients. Among them, the most recently 29 cases were selected for flow cytometry. 9 cases of liver tissues were collected as control group, 6 of them come from the induced labor fetus, and 3 of them come from the patients of neonatal hepatitis.2. Fluorescence quantitative PCRPlates of PCR were prepared by extracting DNA from liver specimens, and the purity of DNA is guaranteed by selecting the value of OD260/OD280 more than 1.8. The FQ-PCR were used to detect HCMV,ADV,EBV,HBV and HSV.3. Pathologic and immunohistochemical stainingThe formalin-fixed paraffin liver specimens were cut into pieces and subsequently performed with pathological and immunohistochemical staining. HE staining, MASSON staining and silver staining were used in pathological staining. Immunohistochemical staining were carried out by using two-step method to dectect IFN-γand HCMV's late protein PP6. DAB was ued for developing color. A clear yellow–brown granular staining could be found when the IFN-γor PP65 was positive in the cytoplasm.4. Flow cytometry detectionSamples of liver tissues were ground and then supernatants were harvested. By Using flowing microsphere technology, 11 kinds of cytokines including: IL-12p70, IFN-γ, IL-2, IL-10, IL-8, IL-6, IL-4, IL- 5, IL-1β, TNF-αand TNF-βwere tested. Final concentrations of each cytokine were calculated by the formula concentration (pg / ml ? mg)=flow results (pg / ml) / weight (mg)×500.5. The culture of human cytomegalovirus culture and its impact on the morphology of human biliary epithelial cell5.1 The culture of human cytomegalovirusHuman embryonic hung fibroblasts were separated and primarily cultured in flask. Then HCMV AD169 was inoculated into the cells, and try to expand it. During the experiment, FQ-PCR were used to monitor the titers of the virus.5.2 The human cytomegalovirus infection on the biliary epithelial cellsHCMV AD169 was inoculated into the biliary epithelial cells monolayers, by every day observation with inverted phase contrast microscope, cells with cytopathic effect were harvested. At the end, FQ-PCR was employed to monitor the virus titers before and after cultivation, and transimission electron microscope (TEM) was used to observe the morphological changes of cells and existence of virus.6. Statistics By using the software of SPSS 13.0, the correlation coefficients of HCMV titers and 11 kinds of cytokines were caculated.Results:1.Distribution of hepatotropic DNA virus in biliary atresia samples The fluorescence quantitative PCR result shows that among 85 cases, 56 cases(65.8%) were found to be positive for hepatotropic DNA virus. Among them, the cases for positive dection of HCMV, ADV, EBV, HSV and HBV were 51(60%), 5(5.9%), 3(3.5%), 0(0%), and 0(0%), respectively. Meanwhile, the biopsies of 3 patients were tested positive for two viruses simultaneously. They include two cases of HCMV in combination with ADV and one case of, ADV in combination with EBV. In the control group, virus detection was negative.2. The pathological changes of biliary atresiaIn the liver tissue of the patients, varying degrees of liver cell degeneration and necrosis, cholestasis in the lobule, even the existence of multinucleated giant cells and Pseudolobule could be found. Some appeared as bile plug formation in the periportal area, bile duct hyperplasia, degeneration and infiltration of lymphocytes and granulocytes, and varying degrees of fibrosis as well. Lymphocytes and granulocyte centered around the bile canalicus, with various degrees of fiber bundle package timid tube.3. The impact of human cytomegalovirus on the morphology of biliary epithelial cells and the distribution of PP65 in biliary atresia tissueSignificant cytopathic effect appeared after inoculating HCMV AD169 into HIBEpiC monolayers. The results of FQ-PCR showed that the viral titer of the supermatants was much higher than 6 days before. By means of TEM, phenomenon of apoptosis was found in the HIBEpiC with viral infection and a large number of viral particles existed in the cells.In all 85 cases we tested, there were 43 cases(52.38%) positive and 42 cases negative, while all of the cases in the control group were negative. PP65 was located in the cytoplasm of infected cells such as liver cells, vascular endothelial cells, bile duct epithelial cells and inflammatory infiltrating cells.4. Results of flow cytometryCompared with the control group, all 11 kinds of cytokines are much higher in the liver tissues of the patients with biliary atresia. Among them, the total amount of Th1 cytokines(IL-1β,IL-2,IL-8,IL-12p70,IFN-γ,TNF-α,TNF-β) and proinflammatory cytokines (IL-1β,IL-2,IL-6,IL-8,IL-12p70,IFN-γ,TNF-α,TNF-β)are much higher than the total amount Th2 cytokine(sIL-10,IL-6,IL-4,IL-5)and the anti-inflammatory factors(IL-4,IL-5,IL-10).5. The expression of IFN-γin the liver tissue of patients with biliary atresia The result showed 35 cases were positive and 23 were negative. IFN-γexpression focused on the portal area, especially in the cytoplasm of inflammatory cells around the bile duct, with proliferation of small bile duct as the center. Biliary epithelial cells were weakly positive for IFN-γstaining.6. The correlation between HCMV titer and 11 kinds of cytokinesAmong 29 cases which carry out the flow cytometry, 15(51.72%)were positive for HCMV, and 9 were negative. The titers of human cytomegalovirus showed different correlations with all 11 kinds of cytokines and strong positive correlation with some kinds of cytokines(IFN-γ,IL-2,IL-10,IL-4,TNF-αand TNF-β), IFN-γis the strongest one(r=0.796). Moreover, it shows strong positive correlation with with the total of Th1 cytokines(r=0.914), proinflammatory cytokines(r=0.913)and the total of all 11 kinds of cytokines(r=0.893).Conclusions:1. Hepatotropic DNA virus has a close relationship with Biliary atresia; Among the hepatotropic DNA viruses we detect, human cytomegalovirus has a large relationship with biliary atresia, it can invade into the biliary epithelial cells and cause the apoptosis of the bile duct epithelial cells;2. The immune reaction of biliary atreisa maybe triggered by the human cytomegalovirus and mostly promoted by the CD4~+ Th1 cells and it's cytokines.