- 學位論文
STAT3在Con A引起肝炎反應中所扮演的角色的研究
The Role of STAT3 in Con A-induced Hepatitis
摘要
STAT3 基因在起始肝臟發育以及再生功能中扮演重要的角色。然而，STAT3 在肝炎反應中的角色仍然不清楚。為了瞭解 STAT3 在 Con A 所引發的肝炎反應中扮演的角色，我們用只剔除肝臟細胞 STAT3 基因（ST3KO）的老鼠來做為動物模式的研究材料。在 Con A 的刺激下，缺少 STAT3 的老鼠血清裡的 ALT 以及 AST 值較正常的老鼠為低，從肝臟切片染色中我們也發現較少的肝臟受損，而且，肝臟細胞的 caspase-3 活性也較正常的老鼠低，另外，這種老鼠在高劑量 Con A 的刺激下所誘發的死亡率也較低，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。 接著我們想進一步研究這種缺少 STAT3 的老鼠抑制 Con A 引發肝炎反應的機制。首先，我們發現在正常老鼠與 ST3KO 老鼠肝臟裡的各種白血球（IHL）比例及數目，如 CD4+ T細胞，自然殺手T （NKT）細胞，顆粒球細胞 (granulocytes) 以及肝臟的巨噬細胞 (Kupffer cells)，在 Con A 刺激前或者刺激後都相當類似；其次， Con A 刺激後血清中促進發炎的細胞激素, 如 IFN-γ、TNF-α、IL-4，以及血清中的抑制發炎的細胞激素，如 IL-10、IL-6 的濃度及肝臟中這些激素的 mRNA 表現量在正常老鼠及 ST3KO 老鼠中都是相似的。這些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，也不會影響細胞激素的產生及釋放。這個推論也由正常老鼠及 ST3KO 老鼠有相似的脾臟受損程度的結果中獲得支持，這個結果進一步證明 ST3KO 老鼠的淋巴球有正常的功能。 為了探討 ST3KO 老鼠在 Con A 模式中有較低的肝炎反應，是否因為缺少 STAT3 的肝細胞可以抵抗細胞激素誘發的細胞死亡。我們將原始的肝細胞 (primary hepatocytes) 以不同的細胞激素去刺激，結果發現 TNF-α/Actinomycin D 誘發的細胞死亡程度在缺少 STAT3 的肝細胞中較低。再者，IL6 造成增強 TNF-α/Actinomycin D 引起的細胞死亡的機制在缺少 STAT3 的肝細胞中則是受到阻礙。 有趣的是，在 Con A 刺激後肝臟裡的 SOCS 被引發的量在 ST3KO 老鼠中是不正常的。在 ST3KO 老鼠的肝臟中， SOCS3 被引發的量遠比正常的老鼠低，而 SOCS1 被引發的量比正常的老鼠高。這些結果暗示，在 ST3KO 老鼠中這些不正常的 SOCS 被引發量可能會影響細胞激素的訊號傳遞，進而抑制 Con A 引發的肝炎反應。 綜合以上的發現，這些結果指出在 Con A 引發的老鼠肝炎動物模式中 STAT3 透過 IL-6-STAT3 的訊號傳遞增強 TNF-α 引起的細胞死亡，因而扮演了正向調控因子的角色，其中一個可能的機制是透過 STAT3 下游的 SOCS1/SOCS3 調控細胞激素的訊號傳遞。
關鍵字
肝炎並列摘要
STAT3 is an essential molecule for the initiation of liver development and regeneration. However the role of STAT3 in liver damage remains unclear. In order to characterize the role of STAT3 in concanavalin A (Con A)-induced model of immune-mediated hepatitis, ST3KO mice were used. After Con A treatment, reduced serum ALT/AST levels, liver injury, apoptosis and high-dose-induced lethality were observed in ST3KO mice as opposed to WT mice, suggesting that STAT3 had a pro-apoptotic role in hepatocytes in Con A-induced hepatitis. The mechanism of ST3KO mice resistant to Con A-induced hepatitis was further studied. First of all, the percentage and numbers of IHLs, including CD4+ T, NKT, granulocytes and kupffer cells were comparable in WT and ST3KO mice before and after Con A treatment. Secondly, mRNA levels in the liver and protein levels in the serum of pro-inflammatory cytokines such as IL-4, IFN-γ and TNF-α and anti-inflammatory cytokines such as IL-6 and IL-10 were similar between WT and ST3KO mice after Con A treatment. These findings suggested that the recruitment of effector cells to liver and the production and release of cytokines were normal in the absence of STAT3. In line with these results, similar severity of pathology was observed in the spleen, where STAT3 was not deleted. These results suggested that lymphocytes of ST3KO mice were functional. To investigate if the impaired Con A-induced hepatitis was due to resistance of cytokine-induced apoptosis in the hepatocytes lacking STAT3, primary hepatocytes were incubated with different apoptosis-inducing cytokines in vitro. Decreased TNF-α/Actinomycin D-induced apoptosis was found in STAT3 knockout hepatocytes. Furthermore, IL-6-mediated enhancement of TNF-α/Actinomycin D-induced apoptosis was completely blocked in the absence of STAT3. Interestingly, the levels of SOCS molecules were dysregulated in the liver of ST3KO mice after Con A treatment. Impaired SOCS3 and prolonged SOCS1 induction were detected in ST3KO mice, implying these atypical SOCS production might be affecting cytokine signaling and contribute to impaired Con A-induced hepatitis in ST3KO mice. Taken together, these results suggested that STAT3 is a positive regulator for Con A-induced hepatitis by regulating the response through IL-6-STAT3 signaling to enhance TNF-α-induced apoptosis. One of the mechanisms is through its downstream targets such as SOCS1/SOCS3 to regulate cytokine responses.