- 學位論文
STAT3在Con A 引起肝炎反應中促進肝細胞進行細胞凋亡的角色研究
The pro-apoptotic role of STAT3 in hepatocytes during Con A-induced hepatitis
摘要
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的動物模式。雖然這一個動物模式已經被沿用了數十年，其反應機制仍然不清楚。為了要研究在Con A所引起肝炎反應中STAT3在肝細胞內的角色，我們用只剔除肝臟細胞STAT3基因(STAT3KO)的老鼠來作為動物模式的研究材料。在我們實驗室之前的研究中發現，STAT3KO老鼠在注射Con A之後，血清中的AST/ALT數值以及肝臟受損程度和肝臟中caspase-3的活性都比正常的老鼠低。在用致死劑量的Con A 注射小鼠時，STAT3KO老鼠則有比較高的存活率。利用體外毒殺試驗 (in vitro killing)，我們進一步確認STAT3在肝臟細胞中的角色，STAT3KO或正常的肝細胞在體外(in vitro)和被Con A 活化過的肝臟內的白血球(IHL)共同培養時，我們發現缺少STAT3的肝細胞比較能抵抗來自正常或STAT3KO肝臟內白血球的毒殺。這些結果皆指出STAT3在Con A 引發的老鼠肝炎的動物模式中扮演促進發炎的角色。 接著我們進一步研究 STAT3KO老鼠抑制Con A引發肝炎的機制。首先，在活體(in vivo)及活體外(in vitro)的實驗中，我們都在STAT3KO老鼠的肝和肝細胞中觀察到比較低的兩種p85α的同種型(isoform) - p55α及p50α的表現。這兩種蛋白質已知會抑制PI3K的活性，並導致較低的細胞存活率。的確，在注射Con A之後，STAT3KO老鼠具有比較高的Akt活性。這些結果顯示STAT3KO老鼠比較不易從p85α轉換成p55α及p50α可能是造成其抑制Con A引發肝炎的原因之一。 為了探討STAT3KO老鼠在Con A 模式中有較低的肝炎反應，是否是因為STAT3KO肝細胞對細胞激素引起的細胞死亡有不同的反應，我們肝細胞 (primary hepatocyte) 在體外以TNF-α/IL-6 刺激，發現誘發細胞死亡的程度在缺少STAT3的肝細胞中較低而且 IL-6造成增強TNF-α引起細胞死亡的機制在缺少STAT3的肝細胞中也是受到抑制。 最後，在利用細胞激素刺激前後，STAT3KO肝細胞比起正常的肝細胞，有較低的SOCS3和較強的SOCS1表現，暗示著在STAT3KO肝細胞中不同的SOCS的表現量可能會影響細胞激素訊息傳遞的路徑進而抑制Con A引發的肝炎反應。 綜合以上發現，這些結果顯示在Con A 引發的老鼠肝炎動物模式中STAT3可能透過促進PI3 kinase 中p85α轉換成p55α及p50α及透過IL-6-STAT3的訊號傳遞增強TNF-α引起的細胞死亡或是經由不同的SOCS產生量來改變細胞激素的訊息傳導這些機制，因而扮演了正向調控的角色。
並列摘要
Concanavalin A (Con A)-induced hepatitis is considered as a model for human fulminant hepatitis. Although this model has been established for decades, the underlying mechanisms are still not fully understood. In order to clarify the role of STAT3 in liver during Con A-induced hepatitis, liver-specific STAT3 conditional knockout mice were used. In our previous studies, reduced serum ALT/AST levels, liver injury and apoptosis were observed in STAT3KO mice after Con A treatment. Higher survival rate was also shown in STAT3KO mice after challenging mice with high-dose Con A. Moreover, STAT3KO hepatocytes were more resistant to in vitro killing of Con A-activated IHLs. These data suggested that STAT3 had a pro-apoptotic role in hepatocytes in Con A-induced hepatitis. The mechanisms of resistance of STAT3KO mice to Con A-induced hepatitis were further investigated. First of all, lower expressions of p55α and p50α, two isoforms of p85α regulatory subunits, were observed in liver and primary hepatocytes of STAT3KO mice both in in vivo and in vitro experiments. Secondly, enhanced Akt activity was observed in STAT3KO mice after administration of Con A. These results suggested that the reduced conversion of PI3 kinase regulatory subunits from p85α to p55α and p50α might contribute to the reduced hepatitis in STAT3KO mice. To investigate if the differential response to cytokine-induced apoptosis also resulted in the resistance of STAT3KO mice in Con A-induced hepatitis, primary hepatocytes were stimulated with TNF-α/IL-6 in vitro. Decreased TNF-α-induced apoptosis was found in STAT3KO hepatocytes and the IL-6-mediated enhancement of TNF-α-induced apoptosis in hepatocytes was not blocked in the absence of STAT3. Finally, impaired SOCS3 and enhanced SOCS1 expression were detected in STAT3KO heaptocytes before and after stimulation, implying the differential SOCS production might affect cytokine signaling pathways and contributed to reduced Con A-induced hepatitis in STAT3KO mice. Taken together, these results suggested that STAT3 is a positive regulator for Con A-induced hepatitis probably by affecting the conversion of p85α to p55α and p50α of PI3 kinase, regulating the response through IL-6-STAT3 signaling to enhance TNF-α-induced apoptosis or altering cytokine responses via atypical SOCS production.