伍安怡臺灣大學：免疫學研究所宋瑞敏Sung, Jui-MinJui-MinSung2007-11-292018-07-092007-11-292018-07-092004http://ntur.lib.ntu.edu.tw//handle/246246/63292在登革感染病人中，可發現淋巴球活化、肝臟內細胞浸潤及肝臟酵素上升的情形，然而卻缺乏合適的動物模型來探討肝臟損傷的機制，本研究中我們使用野生種C57BL/6小鼠，給予靜脈注射DEN-2 (16681)病毒，在登革病毒感染後，發現ALT及AST上升，以及由TUNEL染色在肝臟中所偵測到的凋亡細胞，顯示肝臟的受損，藉由免疫染色的方法，發現感染後第一天浸潤為NK細胞，而給予一次病毒注射後第五天以及給予兩次病毒注射後第三天則是CD4及CD8 T細胞、活化的巨噬細胞以及嗜中性球。分離出肝臟內淋巴球則表現CD44hi的活化表型，而感染後浸潤的CD8+細胞的比率高於CD4+細胞，此種現象在給予兩次病毒注射後較一次注射明顯。 細胞毒性細胞殺標的細胞是藉由perforin及granzyme的路徑，本研究中利用perforin基因剔除小鼠，發現perforin在於肝臟損傷的早期扮演角色，而TCRβ基因剔除小鼠以及在病毒注射前先給予anti-IP10抗體，顯示了NK細胞在肝臟損傷早期的角色，此外，感染後第五天TCRβ基因剔除小鼠的ALT及AST與控制組相同，顯示ㄒ]T細胞在肝臟損傷晚期的重要角色，進一步利用給予anti-CD8抗體以及MHC class I基因剔除小鼠，證明CD8+ T細胞扮演重要的角色，這些發現顯示了NK細胞在早期及ㄒ]T細胞在晚期登革病毒感染小鼠之肝臟損傷中扮演重要的角色。Lymphocyte activation, hepatic infiltration and elevated liver enzyme levels are observed in patients infected with dengue virus. However, the pathogenic mechanism of liver damage has never been studied. In this study, we observed that immunocompetent mice infected with DEN-2 strain 16681 had elevated serum ALT and AST levels and the liver cells were apoptotic, indicating that dengue virus induces liver damage in the mouse. Immunohistochemical staining revealed there was hepatic cellular infiltration and NK cells constituted the majority of infiltrating cells at day 1 of infection. By day 5, infiltrating cells consisted of mostly T cells, macrophages and neutrophils. Flow cytometric analysis revealed greater percentage of CD8 T cells than CD4 T cells in the liver and the ratio was 1.9, and most infiltrating T cells expressed CD44hi phenotype. When mice were given a second inoculation of DEN-2, the CD8 T to CD4 T cell ratio increased to 2.6 at day 3 after the second inoculation. Moreover, the liver enzyme levels also increased in mice after second inoculation, suggesting liver damage is associated with lymphocyte infiltration. TCRβ as well as perforin knockout mice were used in order to delineate the role of T cells and NK cells in mediating liver damage. The results showed that liver enzyme levels in DEN-2 infected TCRβ knockout mice were significantly greater than uninfected controls at days 1 and 3 but not day 5 of infection and the enzyme levels in infected perforin knockout mice were significantly greater in day5, but not days 1 and 3 of infection. Further examination of anti-CD8 antibody treated wild type mice and MHC class I knockout mice indicated the role of CD8+ T cells in liver damage at day 5 of infection. Moreover, treating DEN-2 infected immunocompetent wild-type mice with anti-IP-10 antibody reduced the serum enzyme levels to that as in uninfected mice. These results indicate that a perforin-dependent mechanism, perhaps through recruited NK cells mediated liver damage at early time points of infection, whereas infiltrating T cells mediated liver damage at later time points.Chinese Abstract...…………………………………………............. i English Abstract...………………………………………………….. iii Chapter I. Introduction Dengue virus and dengue………………………………............. 1 Liver damage in dengue infection……………………................ 3 Cellular response to dengue…………………….......................... 3 Cytokine and chemokine response to dengue…………………... 5 Cytotoxic molecules in liver injury……..…………………......... 6 Animal models…………………………………………............. 7 The rationale to study liver injury in dengue virus-infectedmice…………………………………………….......................... 8 Chapter II. Materials and Methods Part 1. Experimental Procedures 1.1 Mice………………………………………………............. 9 1.2 Virus and infection………………………………………... 9 1.3 Cell surface marker staining and flow cytometric analysis…………………………………………………… 10 1.4 Isolation of intrahepatic lymphocytes…………………….. 10 1.5 Immunohistochemistry…………………………………… 11 1.6 TUNEL staining…………………………………………... 11 1.7 Anti-IP10 antibody preparation and treatment……............ 12 1.8 Mouse IgG ELISA………………………………………... 12 1.9 AST and ALT detection…………………………………… 13 Part 2. Experimental Materials…………………………............. 13 Chapter III. Results Liver injury in dengue virus-infected mice…………………….. 20 Identification of immune cells infiltrating the liver…………….. 21 The role of T cells in liver injury……………………………….. 22 The role of IP-10 and NK cells in dengue-virus infected liver tissue……………………………………………................. 24 The role of perforin in causing liver damage…………………... 24 Chapter IV. Discussion Part I. Liver injury in dengue virus-infected mice……………... 26 Part 2. Infiltrating lymphocytes in liver………………………... 28 Part 3. The mechanism of infiltrating cells cause liver damage after dengue virus infection……………….…………………….. 29 Summary………………………………………………………... 32 References.………………………………………………………… 33 Figures Figure 1. Liver enzymes and apoptotic cells in immunocompetent B6 mice after dengue virus infection………………………………………………. 43 Figure 2. Figure 2. CD4+ and CD8+ cells infiltrate into livertissue in immunocompetent B6 mice after denguevirus infection.………………………………………... 45 Figure 3. Marcophages and neutrophils infiltrate into liver tissue in immunocompetent B6 mice after dengue virus infection. 47 Figure 4. Expression of CD44hi in CD4+ and CD8+ cells and the percentage of lymphocyte gate of CD4+ and CD8+ cells increased after dengue virus injection…………... 49 Figure 5. Liver enzymes and apoptotic cells in TCRb knockout mice after dengue virus infection…………………….. 52 Figure 6. No liver cells undergo apoptosis with CD8+ T celldepletion……………………………………………… 54 Figure 7. No liver cells from MHC class I knockout mice undergo apoptosis after dengue virus injection………. 55 Figure 8. Decreased natural killer cells infiltrated in liver afterdengue virus injection with anti-IP10 treatment……….. 56 Figure 9. Serum AST and ALT level of B6 mice with or withoutanti-IP-10 treatment prior dengue virus infection d1… 57 Figure 10.Liver enzymes and apoptotic cells in perforin knockout mice after dengue virus infection…………….. 58en-US細胞浸潤肝臟損傷登革熱病毒liver injurycellular infiltrationdengue virus[SDGs]SDG3other