The Investigation Of Mechanism For Inadequate Regulation Of Hepatic NK Cells On Mφ Cells Induceing Biliary Atresia In Newborn Mice

Part Ⅰ. After the mouse were challenged with RRV at different age, there were distinction of activation of hepatic NK cells.AbstractObjective: To confirm the distinction of NK activation from livers after the mouse were challenged with RRV at different age.Methods:After the mouse were infected by RRV at1day old or the Adult for24hours, we isolated CD49+/CD5-NK cells by magenetic activated cell sorting (MACS) from the livers of1day old or the Adult. We defined activated NK cells as those which were positive for CD69, TNF-a and IFN-y. Then, Flow cytometric analysis showed the distinction of the expression of CD69, TNF-a and IFN-y in these NK cells.Results:Comparing with the neonatal mouse without RRV challenging, the NK cells from the neonatal mouse challenged with RRV expressed higher CD69, TNF-a and IFN-y, p<0.05, with satistically signigicant. As well, the NK cells from the adult challenged with RRV expressed higher CD69, TNF-a and IFN-y, too, p<0.05, with satistically signigicant. But, the NK cells from neonatal mouse challenged with RRV had less expression than the Adult with RRV challenging, p<0.05, with satistically signigicant.Conclusions:This study showed that RRV could induce activation of NK cells in mouse liver, but the ability to activate NK cells in the liver of neonatal mose was significantly lower than in adult mice. With neonatal mouse age, the neonatal mouse liver NK cells mature, and their ability to activate gradually increased. Part Ⅱ. Isolation and Identification of phagocytic activity methods for Kupffer cells from neonatal mouseAbstractObjective: To establish a convenient method of isolating Kupffer cells from neonatal mouse liver cells and to provide reliable experimental cells for investigating the pathogenesis of Biliary Atresia.Methods: A parenchymal hepatocyte enriched suspension was prepared from6-8neonatal mouse liver cells and seeded into culture flasks. After3to4days of culture, when most hepatocytes were vigorously proliferated on the cell sheet. By cell scraper, the adherence cells were readily detached and transferred in another plastic dishes. After45minutes incubation, the quick and selective adhesion cells were what we wanted:second selective adherence cells which were identified by immunofluorescence method (IFA) of F4/80. Incubated with light microscopy(2um) for1hour, these second adherence cells were observed under the laser confocal microscope.Results:These second adherence cells were identified by their immunoreactivity to the F4/80antibody, and to99%purity. As more than107F4/80positive cells could be recovered simply from the method. The cells possessed active phagocytosis of typical macrophage as light microscopy (2um) could be seen in the cytoplasm.Conclusions: Our procedure might implicate a novel alternative by selective adhesion to obtain Kupffer cells from neonatal mouse liver cells in sufficient number and purity without complex equipment and skills, which is especially usefully for studying the pathogenesis of Biliary Atresia. Part Ⅲ. Dependent on IL-4and IFN-y, hepatic NK of mouse in different ages had diverse adjustion on Mφ.AbstractObjective:there was insufficient in activation ability of hepatic NK cells from newborn mice, and activated NK cells were the major source of cytokines IFN-y and IL-4. We hepothesised that there was dose dpendence of Mcp cells activation throug specific cytokine IFN-γ and IL-4, and then clear the activated hepatic NK cells of neonatal mouse were not enough for regulating Mcp cells activation.Methods: IFN-y could assist Mcp cells activation for improving cytokine expression of iNOS and TNF-a. Mcp cells were incubated with different doses of IFN-y for12hours, by westblot and real-time PCR to detect the relationship between expression and transcription of cytokines TNF-a and iNOS and dose of IFN-y; and through ELISA assay and RT-PCR to detect the relationship between expression and transcription of cytokine IL-10and dose of IL-4after Mcp cells were incubated with different doses of IL-4for12hours. Mφ cells were from neonatal mouse and adult muose, respectively.Results:After Mcp cells incubation with different doses of IFN-y for12hours, the expression and transcription of cytokines iNOS and TNF-a showed an increasing trend with increasing doses of IFN-y. Similarly, with increasing concentrations of IL-4, the expression and transcription of cytokine IL-10also gradually increased, but when the concentration of IL-4reached to100u/L, the expression and transcription of cytokine of IL-10reached a peak and then gradually decreased. Activated Mcp cells had the same trend, whatever from neonatal mouse liver or adult mouse liver. Conclusions: The manner of activation of Mcp cells was through cytokine IFN-y and IL-4dose-dependent manner. Within a certain range, with the dose of the cytokines IFN-y and IL-4incubated with them increased, their ability to secrete inflammatory cytokines went up. And the activated NK cells are the major source of the cytokines IFN-y and IL-4, but there was insufficient in activation ability of hepatic NK cells from newborn mice. And then it cleared the hepatic NK cells of mice of neonatal mouse were not enough in the regulation of activatio of Mcp cells. Part IV. For Mφ cells which engulf RRV, there was different ability of killing of hepatic NK cells from mouse at different ages.AbstractObjective: To compare the difference in the ability of hepatic NK cells of mouse at different ages to kill Mφ cell which engulfed the RRV, which further determined the hepatic NK cells in newborn mice is insufficient in regulating Mφ cells.Methods: By magnetic cells sorting and selective adhesion, we abtained hepatic NK and Mφ cells from wild-type B6mouse at different ages, respectively. Depending on hepatic NK cells from mouse at different ages and whether NK cells activated by cytokine HMGB1in advance, the NK cells were divided into A, B, C, D four groups. After the NK cells cultured with Mφ cells of mouse at different age which engulfed the RRV for4hours, we dectected the ability of the NK cells of different group Kiling the Mφ cells by a non-radioactive cytotoxicity assay.Results:The ability of neonatal mouse hepatic NK cells activated by cytokine HMGB1to kill the hepatic Mφ cells of mouse at different age which engulfed RRV was significantly enhanced than the NK cells which not activated, P<0.05, statistically significant; Similarly the ability of adult mouse hepatic NK cells activated by cytokine HMGB1to kill the hepatic Mφ cells of mouse at different age which engulfed RRV was significantly enhanced, too, P<0.05, statistically significant. However, compared with the cytotoxic activity of the adult hepatic NK cells, the cytotoxic activity of the hepatic NK cells of neonatal mouse was obviously insufficient, P<0.05, statistically significant. But with increasing age in mice, NK cells mature and the cytotoxic activity of NK cells gradually increased.Conclusion: The ability of neonatal mouse hepatic NK cells to kill the Mφ cells which engulfed RRV was insufficent, and they could not to effectively remove Mφ cells which engulfed. So, this leaded to RRV persist and multiply in the body and caused a lot of damage of biliary epithelial cells and chronic inflammation, followed by the formation of biliary atresia...