李建國臺灣大學：免疫學研究所關珞Kuan, LoLoKuan2007-11-292018-07-092007-11-292018-07-092004http://ntur.lib.ntu.edu.tw//handle/246246/63332利用Con A 注射在小鼠引發嚴重肝臟病變是研究人類肝炎的一個很好動物模式。即使這個動物模式已經發展了數十年，但是它真正引發肝病變的機制仍未清楚。在此我們利用傳統的STAT1基因剔除鼠（ST1KO）以及只剔除肝臟細胞的STAT3基因（ST3KO）的老鼠來研究STAT1和STAT3在Con A引發的肝炎模式中所扮演的角色。 Con A的刺激下正常老鼠肝臟細胞中的STAT1以及STAT3都會短暫的被活化。雖然，STAT3被活化的程度在ST1KO的老鼠以及正常的老鼠是沒有太大的差別，然而STAT1在ST3KO的老鼠裡活化的程度卻比正常的老鼠高。有趣的是，在 Con A的刺激下在缺少STAT1或者STAT3的老鼠中都只產生輕微的肝臟病變，這兩種老鼠血清裡的ALT 以及AST都較正常的老鼠為低，從肝臟切片中我們也發現沒有或較少的肝臟受損，或者肝臟細胞的caspase 3活性較正常的老鼠低。 接著我們想進一步研究這兩種mutant老鼠抵抗Con A引發肝炎的機制。首先，我們發現在正常老鼠與ST3KO老鼠中肝臟裡的各種淋巴球（IHL）數目，如natural killer T（NKT）細胞，CD4+ T細胞，以及顆粒球細胞，在Con A刺激前或者刺激後都相當類似，此結果暗示肝臟細胞的STAT3被剔除後並不影響effector細胞被徵召回肝臟中。第二，Con A刺激後血清中促進發炎的細胞激素知, 如IFN-Concanavalin A (Con A)-induced severe liver damage in mice is considered a model for human hepatitis. Although this model has been established for decades, the underlying mechanisms are still not fully understood. By using STAT1 traditional knockout mice and conditional knockout mice lacking STAT3 in the liver, the roles of STAT1 and STAT3, two important transcriptional factors, in Con A-induced hepatitis are investigated. Both STAT1 and STAT3 are transiently activated in the hepatocytes of wild-type (WT) mice by tyrosine phosphorylation following Con A administration. While levels of STAT3 activation in STAT1 knockout (ST1KO) mice are comparable to that of Wild-type (WT) mice, STAT1 activation, however, is enhanced in STAT3 conditional knockout (ST3KO) mice. Interestingly, Con A-induced hepatitis is greatly impaired in mice either lacking STAT1 or STAT3 in the liver, with reduced serum alanine transaminase (ALT) and asparate transaminase (AST), less severe liver damage in histological sections, and decreased caspase-3 activity in the Con A-treated hepatocytes. The mechanisms of mutant mice resistant to Con A-induced hepatitis are further explored. First of all, the number of intrahepatic leukocytes (IHLs), including natural killer T (NKT), CD4+ T cells, and granulocytes are found to be similar between WT and ST3KO mice before and after Con A treatment, suggesting that the recruitment of these effector cells to liver is normal in the absence of STAT3. Secondly, profiles of serum pro-inflammatory cytokines such as IFN-誌謝…………………………………………………………………….. iii 中文摘要……………………………………………………………… iv Abstract………………………………………………………………… vi Table of Contents……………………………………………………. viii List of Tables and Figures…………………………………………. xi List of Abbreviations………………………………………………. xiii Chapter I Introduction…………………………………................. 1 1.1 T cell mediated hepatitis inducing by Con A………………………… 1 1.2 JAK-STATs signaling pathway………………………………………. 2 1.3 SOCSs……………………………………………………………..… 3 1.4 Interaction between STAT1, STAT3, and Con A-induced hepatitis….. 4 1.5 Hepatic NKT cells in Con A-induced hepatitis…………………….... 5 1.6 Significance……………………………………………………….… 6 1.7 Specific aims………………………………………………………… 6 Chapter II Materials and Methods……………………………... 7 2.1 Mice………………………………………………………………… 7 2.2 Induction of mouse hepatitis………………………………………... 8 2.3 Measurement of ALT and AST activities…………………………... 8 2.4 H&E staining of liver sections……………………………………… 8 2.5 Caspase-3 activity assay…………………………………………….. 8 2.6 Western Blot………………………………………………………… 9 2.7 RT-PCR……………………………………………………………... 9 2.8 Isolation of mouse intrahepatic leukocytes…………………………. 10 2.9 Flow cytometry analysis of intrahepatic leukocytes………………... 11 2.10 Cytometric Bead Assay (CBA)……………………………………. 11 Chapter III Results………………………………………………… 12 3.1 JAKs, STATs, and cytokines involved in Con A-induced hepatitis.... 12 3.2 STAT1 and STAT3 are activated after Con A treatment………......... 12 3.3 Imapaired elevation ALT and AST levels in ST3KO and ST1KO mice after Con A treatment……………………………………………... 13 3.4 Decreased liver injury in ST3KO mice and no liver injury in ST1KO mice after Con A treatment……………………………………. 14 3.5 Reduced apoptotis in the liver of ST3KO or ST1KO mice after Con A treatment……………………………………………........................... 15 3.6 Comparable number and percentage of different subsets of IHLs in the liver effector cells in WT and ST3KO mice after Con A treatment... 16 3.7 Comparable levels of pro-inflammatory cytokines produced in WT and ST3KO mice after Con A treatment……………………………….. 19 3.8 Comparable liver mRNA of Fas, DAPK-1, and DAPK-2 expression levels in WT, ST3KO, and ST1KO mice before and after Con A treatment………………………………………………………... 21 3.9 Induction of SOCS1 and SOCS3 in the liver is impaired in ST1KO and ST3KO mice, respectively, in response to Con A treatment………. 22 Chapter IV Discussion……………………………………….......... 24 4.1 Con A-induced hepatitis in ST3KO and ST1KO mice…………….. 24 4.2 Evidence support protection of ST3KO mice in Con A-induced Hepatitis…………………………............................................................ 25 4.3 Mechanism of suppression of Con A-induced hepatitis in ST1KO mice……………………........................................................................... 27 4.4 Pro-apoptotic molecules in Con A-induced hepatitis………………. 28 Reference…………………………………………………………….. 30 List of Tables and Figures Table 1. Cytokines, JAKs and STATs that are involved in Con A-induced hepatitis……………………………………………………... 37 Figure 1. STAT1 and STAT3 are activated in the liver by Con A treatment………………………………………………………………… 38 Figure 2 Reduced elevation of serum ALT and AST in ST1KO and ST3KO mice.…………………………………………………………… 39 Figure 3. Reduced elevation of serum ALT and AST in ST1KO and ST3KO mice.……………………............................................................ 40 Figure 4. Reduced liver damage in ST3KO and ST1KO mice……….... 41 Figure 5. Reduced liver damage in ST3KO and ST1KO mice………… 42 Figure 6. Reduced caspase-3 activity in the liver of ST3KO or ST1KO mice after Con A treatment.……………….............................................. 43 Figure 7. Increased proportion of CD4+ and CD8+ cells in the liver and spleen in ST1KO mice compared to WT and ST3KO mice.…………… 44 Figure 8. Comparable IHL yields in WT and ST3KO mice…………… 45 Figure 9. NKT cells are greatly reduced in WT, ST1KO, and ST3KO mice after Con A treatment.…………………………………………….. 46 Figure 10. Comparable percentage of CD3+ T cells in WT and ST3KO mice after Con A treatment.…………………………………………….. 47 Figure 11. Comparable percentage of CD4+ and CD8+ T cells in WT and ST3KO mice after Con A treatment.…………………….................. 48 Figure 12. Comparable percentage of NK and CD11b+ cells in WT and ST3KO mice after Con A treatment.…………………………………… 49 Figure 13. Comparable percentage of granulocytes and B cells in WT and ST3KO mice after Con A treatment.……………………………….. 50 Figure 14. Profiles of serum IFN-573009 bytesapplication/pdfen-US肝炎模式訊息傳遞發炎肝臟SOCS1NKTIHLSTAT3STAT1ALTCon A-induced hepatitisSOCS3AST[SDGs]SDG3otherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/63332/1/ntu-93-R91449007-1.pdf