| OBJECTIVE: Hepatitis B virus (HBV) infection has been a worldwide public health problem. Hepatitis B vaccine can prevent occurrence of Hepatitis B (HB), and can achieve satisfactory results for control of intrapartum and postpartum HBV transmission. But, it could not exert any effects on control of HBV intrauterine transmission (infection of fetus through placentae), even together with other approaches. Therefore, along with the universal application of efficient vaccines to hepatitis B, studies on HBV intrauterine transmission have been one of the key points for control of hepatitis B prevalence. Although the pathogenesis of HBV transmission to the fetus during pregnancy is unknown, congenital HBV infections are commonly associated with infection of the placenta. Thus, previousrelative in vivo and epidemiological studies on HBV intrauterine transmission were focused on the placenta. Analyzing the results derived from experimental studies and field studies comprehensively, the researchers suggest that HBV intrauterine transmission may occur either through placental tears with transfusion of infected maternal blood into the fetal circulation, which is the named "hematogenous transmission", or through progressive infection of different placental layers until the virus reaches the fetoplacental circulation, which is the named "transplacental cellular traffic". But the exact mechanism of transmission has yet to be defined. To test the hypothesis derived from the in vivo studies, and to probe thoroughly the exact mode through which HBV entered the placenta barrier cells, approapriate in vitro experimental system is necessary. Trophoblast cells, as the outermost covering of placental barrier, are directly bathed in the maternal blood, might be the key sites for invasion of HBV into placentae. Choriocarcinoma cells, which derive from the trophoblast layer, share numerous properties with trophoblast cells and provide an ideal monocellular system with which to study virus infection of the placental barrier.Besides, the physiological activities are complicated and unique during pregnancy. Numerous extra-cellular soluble factors are present in the vicinity of the placenta during gestation [interleukin (IL)-1,3,4,6, granulocyte-macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), transforming growth factor (TGF)- β , interferon (IFN)-γ , tumor necrosis factor (TNF-)α ,epithelial growth factor (EGF), vascular epithelial growth factor (VEGF), progesterone and estrogen]. These agents play pivotal roles during gestation and are mandatory for a successful pregnancy. Recentaly, roles of tumor necrosis factor (TNF-) a in virus intrauterine transmission have drawn great attention from researchers. Studies suggested that the presence of cytokine TNF- a in the vicinity of trophoblastic cells would create more favorable conditions leading to vertical transmission of virus. We aimed to establish an in vitro experimental model that mimics the interaction of HBV and the trophoblastic barrier based on close parallel to physiological conditions, to provide solid cellular basis for studies on mechanism of HBV intrauterine transmission.MATERIALS AND METHODS: JAR cells, obtained from Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy Of Sciences, were grown in RPMI1640 supplemented with 10% fetal bovine serum and antibiotics (penicillin 100 units/ml, and streptomycin 100 u g/ml). Cells were seeded at a concentration of 2X105 cells per ml in flasks. The medium was changed every 2 days. Cultures were observed daily using a phase-contrast microscope. At confluency, cell monolayers were trypsinized and passaged, or frozen in liquid nitrogen till they were enough for the subsequent experimental manipulation. The sera containing approximately 2X109DNA genome equivalents per ml were obtained from a patient with envelope antigen HBeAg-positive chronic hepatitis B, who had elevated alanine aminotransferase levels(>100UI/ml), divided into aliquots, and stored at -70°Cfor future use. All virus preparations underwent only one freeze-thaw cycle before initiation of infection studies. The virus stock was diluted prior to inoculation into prewarmed (room temperature) culture medium. When JAR cells reached a 50%-80% confluence, 2 to 6X 106 of JAR cells were incubated with 2 to 6X108 of HBV (corresponding to approximately 100 viral genomes equivalent/cell) in the presence (+TNF a )Or absence (-TNF a ) of TNF a (at a final concentration of 10 ng/ml) at 37°C under a 5% CO2 atmosphere to allow for virus uptake. After an overnight incubation, cells were then washed three times with PBS, trypsinized, extensively washed till the last wash was negative for HBsAg by ELISA. Thus, viruses weakly associated with the cells were eluted by trypsinization and extensive washings. The cells were subcultured in culture flasks and coverslips simultaneously in complete RPMI1640 culture medium. At 12 h intervals, the cells and culture media were harvested for subsequent detection. Daily visual inspection and trypan blue antistaining of the cell cultures revealed no evidence of a cytotoxic effect. The infection efficiency was examined by ELISA of culture supernatants, immunohistochemistry of cell slides, Western blotting of cell lysates, PCR amplification for viral DNA of infected cells, and transmission electron microscopy for HBsAg particles in infected cells. RESULTS: Culture medium was collected at hours 36,48, 60, 72, 84, 96 after infection (at hours 12, 24, 36, 48, 60, 72 after subculture). Secretion of HBsAg was observed at hour 36 post-infection. Theproduction of HBsAg by +TNF a JAR cells was significantly higher than that by -TNF a JAR cells. The differences observed between the various time points did not reach a statistically significant level. By immunocytochemistry, we found positive or strong positive staining for HBsAg in HBV-infected JAR cells in the presence of TNF a and negative or weak positive staining in HBV-infected JAR cells in the absence of TNF a . The staining was significantly different between +TNFa JAR cells and —TNF a JAR cells (6.40 + 2.07 vs 20.00 + 5.70, P< 0.01). +TNFa JAR cells exhibited strong reactivity for anti-HBs. Both membrane and cytoplasmic localization of HBsAg were observed. The frequency of HBsAg positive cells at different time points did not show any significant changes. At various time points, an approximately 522 bp DNA band was detected. But in the HBV infected JAR cells which were not pretreated with TNF a , no or very light specific band of PreS cDNA was observed. HBV infected JAR cells pretreated with TNF- a repeatedly gave a positive intracellular HBsAg signal by Western blotting assay.And electron microscopic examination revealed the presence of band-form HBsAg particles in dilated rough surfaced endoplasmic reticulum cavities. CONCLUSION: The results suggest that trophoblast-derived choriocarcinoma cells are sensitive to infection with HBV in vitro. In addition, we have found that infection of trophoblast cells by HBV was enhanced in the presence of tumour necrosis factor alpha, which mimics the physiological conditions of trophoblasts closely during pregnancy. We carried out the in vitro study on HBV intrauterine... |
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