李建國Lee, Chien-Kuo臺灣大學:免疫學研究所李靜蓉Lee, PriscillaPriscillaLee2010-05-102018-07-092010-05-102018-07-092008U0001-0608200815461400http://ntur.lib.ntu.edu.tw//handle/246246/181783利用在小鼠體內注射入Concanavalin A (Con A)所引起之嚴重肝臟病變是一個用來研究人類猛暴性肝炎或自體免疫引起之肝炎的很好的動物模式，但其致病機轉至今仍尚未完全明朗。為了釐清在Con A 引起之肝炎中STAT3 在肝細胞內扮演的角色，我們使用只在肝細胞中剔除STAT3基因(STAT3KO)的小鼠作為通篇研究的實驗材料。根據先前的研究，我們發現在Con A刺激下STAT3KO小鼠血清中的ALT/AST數值以及肝臟受損程度相較於正常小鼠都是較低的。利用體外培養的系統，我們現在發現STAT3KO的肝細胞比正常的肝細胞更能抵抗浸潤在肝臟中之白血球的毒殺，並且STAT3KO的肝細胞在TNF-α and Fas-agonist的處理下，較不容易進行細胞凋亡。這些結果都顯示著STAT3能夠正向調控在活體中Con A引起或是在體外培養系統由death receptors所誘發之肝細胞的細胞凋亡。著，我們進一步探討STAT3KO 小鼠抵抗Con A引起肝炎之機制。在活體實驗中以Con A處理小鼠的前後，我們觀察到death receptors例如TNFR and Fas的表現量在STAT3KO的小鼠與正常的小鼠中是相似的。兩個已知的anti-apoptotic基因並且曾經被報導過是STAT3下游基因—Bcl-2與Bcl-XL的表現量在Con A處理過後在STAT3KO的老鼠及正常的老鼠中也是相似的。除此之外，我們更觀察到了在Con A處理過後，interleukin-15 and peroxiredoxin2這兩個基因在STAT3KO小鼠中的表現量相較於正常小鼠都有顯著的增加，但仍須後續實驗來證實STAT3KO的小鼠較能夠抵抗Con A引起之肝炎是由於這兩個基因表現量增加所導致。注射D-galactosamine (D-gal) 加上LPS會在小鼠體內引起發炎性急性肝炎，並且也是一個廣泛用來研究人類急性肝炎的動物模式。最後，我們利用D-gal 加上LPS刺激小鼠，並且觀察到STAT3KO的小鼠同樣地能夠抵抗D-gal 加上LPS所引起之肝炎反應。結以上，我們的發現顯示在發炎性急性肝炎中，STAT3在肝細胞中是一個能夠促進細胞凋亡的分子。Concanavalin A (Con A)- induced hepatitis is a well established animal model for studying human fulminant and autoimmune hepatitis. However, the underlying mechanism is not fully understood. To clarify the role of STAT3 in Con A-induced hepatitis in hepatocyte, liver-specific STAT3 conditional knockout mice were used in our research. We previously showed that reduced serum ALT/AST and mild liver damage were observed in STAT3KO mice in response to Con A treatment. We now show that, STAT3KO hepatocytes are more resistant to the killing of Con A-activated IHLs in vitro. STAT3KO hepatocytes are also more resistant to TNF-α and Fas-agonist induced apoptosis of hepatocytes in vitro. These results suggest that STAT3 positively regulates Con A and death receptor-induced apoptotic pathway in hepatocytes in vivo and in vitro.he mechanisms of resistance of STAT3KO mice to Con A-mediated hepatitis are further investigated. While comparable levels of death receptors such as TNFR and Fas are expressed in hepatocytes of WT and STAT3KO mice before and after Con A treatment, the expression of Bcl-2 and Bcl-XL, two anti-apoptotic genes, that have been reported as STAT3 downstream gene, is also similar between WT and STAT3KO hepatocytes after treatment. Furthermore, significantly increased levels of interleukin-15 and peroxiredoxin2 are observed in STAT3KO hepatocytes when compared to WT control. Further investigation is needed to characterize the contribution of these two moleculesto resistant phenotype of STAT3KO mice during Con A-mediated hepatitis. A similar phenotype is seen when mice are injected D-galactosamine plus LPS which is also a well-known animal model for acute hepatitis. aken together, our findings suggested that STAT3 is a pro-apoptotic molecule in hepatocytes during acute inflammatory hepatitis.誌謝………………………………………………i文摘要………………………………………………iibstract………………………………………………ivbbreviation……………………………………………viable of contents………………………………viiiist of tables and figures………………………………………………xhapter I Introduction…………………………………………1.1 Concanavalin A-induced hepatitis………………………………1.2 JAK-STAT signaling pathway…………………………………1.3 Physiological function of STAT3………………………2.4 IL-6-STAT3 signaling pathway………………………3.5 IL-15…………………………………………………3hapter II Materials and Methods……………………………5.1 Mice……………………………………………………5.2 Genotyping of targeted alleles………………………5.3 Induction of mouse hepatitis with Con A or LPS plus D-gal…………6.4 Hematoxylin-eosin (H&E) staining of liver sections…7.5 Measurement of ALT and AST…………7.6 Isolation of mouse intrahepatic leukocytes (IHLs)………7.7 Preparation of primary mouse hepatocytes……………8.8 Isolation of splenocytes…………………9.9 Flow cytometry analysis…………………9.10 Caspase-3 activity assay………………………10.11 In vitro killing assay…………………………11.12 In vitro apoptotic assay ……………………………11.13 Western blotting………………………………12.14 Isolation of RNA, preparation of cDNA and QPCR…12.15 Single cell PCR………………………………15.16 ELISA……………………………………16.17 Microarray assay………………16hapter III Results…………………………………17.1 Reduced serum of ALT and AST in STAT3KO mice after Con A treatment………17.2 Decreased liver injury in STAT3KO mice after Con A treatment………17.3 Comparable percentage of different subsets of intrahepatic lymphocytes in WT and STAT3KO mice before and after Con A treatment…………………18.4 Comparable activation status of IHLs in WT and STAT3KO mice…19.5 STAT3KO hepatocytes were more resistant to in vitro killing of Con A-activated IHLs………………20.6 IL-6 mediated enhancement of TNF-α-induced apoptosis in hepatocytes is STAT3-dependent………………22.7 The proapoptotic role of STAT3 was cell autonomous……23 .8 Fas-mediated apoptosis is more severe in STAT3KO primary hepatocytes……………24.9 STAT3 did not affect intrinsic pathway-induced apoptosis……………25.10 Gene profiles of WT and STAT3KO hepatocytes after Con A treatment……………………25.11 STAT3KO mice were also resistant to D-gal plus LPS-induced hepatitis………………27hapter IV Discussion…………28.1 Pro-apoptotic role but not anti-apoptotic role of STAT3 in hepatocytes during acute inflammatory hepatitis…………………………28.2 Enhanced expression of IL-15 and Prdx2 in STAT3KO mice after Con A treatment…………………………32eferences………………………………………33ables and Figures……………………………………………38able 1 Microarray analysis……………………………39igure 1 Reduced serum ALT and AST levels in STAT3KO mice after Con A treatment……………………………42igure 2 Reduced liver damage in STAT3KO mice after Con A treatment…………43igure 3 Comparable percentage of different subsets of IHLs in WT and STAT3KO mice before and after Con A treatment………………………………44igure 4 Comparable percentage of different subsets of splenocytes in WT and STAT3KO mice before and after Con A treatment………………………………45igure 5 The percentage of CD69 positive cells in each subsets of cells was similar between WT and STAT3KO mice after Con A treatment………………46igure 6 STAT3KO hepatocytes were more resistant to killing of Con A-activated IHLs in vitro…………………………………………47igure 7 Enhancement of TNF-α-induced apoptosis by IL-6 is STAT3-dependent…48igure 8 STAT3-deleted hepatocytes are more resistant to TNF-α-induced apoptosis………………………………………49igure 9 STAT3KO hepatocytes are more resistant to Fas agonist-induced apoptosis………………………………………50igure 10 Comparable UV-induced apoptosis between WT and STAT3KO hepatocytes………………………………………………51igure 11 Comparable levels of pro-inflammatory cytokines and death receptors in hepatocytes of WT and STAT3KO mice after Con A treatment………………………52igure 12 Expression of STAT1 or STAT3 inducible gene in hepatocytes of WT or STAT3KO mice after Con A treatment………………………………53igure 13 Expression of anti-apoptotic genes and ROS-regulated genes in hepatocytes of WT or STATKO mice after Con A treatment………………………54igure 14 Enhanced expression of IL-15 and Prdx2 in STAT3KO mice after con A treatment………………………………55igure 15 Reduced liver damage and splenic hemorrhage in STAT3KO mice in response to D-gal plus LPS…………………………………………56igure 16 Reduced production of proinflammatory cytokines in STAT3KO mice in response to D-gal plus LPS…………………………………………57application/pdf11438989 bytesapplication/pdfen-USSTAT3concanavalin A急性肝炎細胞凋亡hepatitisapoptosis[SDGs]SDG3http://ntur.lib.ntu.edu.tw/bitstream/246246/181783/1/ntu-97-R95449010-1.pdf