| 摘要: | 當肝臟受到生理性或病理性刺激時，肝細胞會利用各種訊號傳導路徑引起凋亡的發生。而參與肝細胞凋亡的因子包括肝臟自主神經系統及細胞激素；在細胞激素（Cytokine）方面，如腫瘤壞死因子（Tumor necrosis factor；TNF-α），當TNF-α與肝細胞上的接受器相結合後會活化下游的Caspase-3，即是誘導肝細胞產生凋亡的路徑之一；在自主神經方面，交感神經系統會抑制細胞凋亡和促進肝細胞的生長，而副交感神經會在下視丘腹側核受損時促進肝細胞的凋亡。 本研究之目的在以自主神經抑制下合併四氯化碳誘導急性肝損傷的動物模型中，探討在細胞凋亡時自主神經系統、TNF-α與Caspase-3之間相互的關係。實驗分組包括：（A）正常對照組、（B）四氯化碳組、（C）假手術切除迷走神經+四氯化碳組、（D）手術切除迷走神經（Vagotomy）+四氯化碳組、（E）化學性阻斷副交感神經（Atropine）+四氯化碳組、（F）交感神經破壞（6-OHDA）+四氯化碳組。藉由H&E染色觀察肝組織病理形態變化，以TUNEL方法觀察肝細胞凋亡出現的情形，並以反轉錄聚合反應及免疫組織化學染色，檢測肝臟組織TNF-α和Caspase-3的表現。 H&E染色之結果顯示，在自主神經抑制下合併四氯化碳誘導急性肝損傷（B、C、D、E、F組）的組織中，其肝細胞可觀察到脂肪變性，但各組間無明顯差異；利用TUNEL方法可於各組肝組織中央靜脈周圍偵測到凋亡細胞，與四氯化碳組（B、C組）相比，於交感神經破壞組（F組）的凋亡細胞數呈現增加的趨勢，而於副交感神經抑制組（D、E組）的凋亡細胞數則是呈現下降的情況；免疫組織化學染色法分析活化的Caspase-3與TNF-α，可於（B、C、D、E、F組）肝組織中央靜脈周圍觀察到活化的Caspase-3陽性細胞表現，另一方面，可於各組肝臟組織血管內皮細胞、庫氏細胞與部份肝細胞上觀察TNF-α陽性細胞表現，各組活化的Caspase-3與TNF-α陽性細胞數表現的結果與TUNEL陽性細胞數相符，為交感神經破壞組（F組）較四氯化碳組（B、C組）多，而副交感神經抑制組（D、E組），較四氯化碳組（B、C組）少。 根據結果得知：四氯化碳會藉由TNF-α導致細胞凋亡，而在交感神經破壞下會導致TNF-α、Caspase-3與細胞凋亡的表現增加，相反地，當副交感神經抑制時，會使TNF-α、Caspase-3與細胞凋亡的表現下降。因此推論，自主神經可能藉由直接調節TNF-α的表現，進而影響到Caspase-3的活化，達到調控在四氯化碳誘導急性肝傷害下所產生的細胞凋亡。; Hepatic apoptosis occurs when the liver is stimulated in either physiological or pathological condition through several cellular signaling pathways. Two factors, autonomic nerve system and tumor necrosis factor-α (TNF-α), play important roles in liver apoptosis process. TNF-α can activate Caspase-3 that would induce hepatocytes apoptosis in acute damaged liver tissue. Another potent factor is autonomic nerve system. Sympathetic nerve regulates liver regeneration and apoptosis. Parasympathetic nerve would stimulate apoptosis in liver after ventromedial hypothalamic(VMH) lesion. This study aims to find out the roles of ANS, TNF-α and Caspase-3 in apoptosis after CCl4 induced acute liver damage rats. Sympathetic inhibition, 6-hydroxydopamine(6-OHDA), or parasympathetic nerve, including vagotomy(VGX) and atropine injection, are performed respectively and followed CCl4 administration. 56 rats are divided into 6 groups:(A) Normal, (B) CCl4, (C) Sham operation+ CCl4, (D) VGX+ CCl4, (E) Atropine+ CCl4, and (F) 6-OHDA+ CCl4. The histological observation in HE stain shows hepatocyte fatty change at 24 hours after CCl4 administration in B,C,D,E,F groups. Based on the TUNEL assay, apoptosis is mainly detected in hepatocytes around the centrilobular zone and the number is increased in the sympathetic nerve inhibition group (F) when compared with the CCl4 group (B and C). On the other hand, parasympathetic nerve inhibited rats, groups D and E, show fewer apoptosis cells than those CCl4 damaged rats. Immunohistochemical stains and RT-PCR are used to clarify the relationship between TNF-α and Caspase-3. TNF-α and Caspase-3 express after acute liver damage. TNF-α expresses in the vascular endothelial cells, Kupffer cells as well as few hepatocytes and Caspase-3 signals exhibit in hepatocytes around central vein. The positive cells are counted and the numbers increase in group F but decrease in group D and E. These results coincide with the TUNEL assay. In this study, apoptosis in rat liver could be induced by CCl4 administration through the TNF-α/ Caspase-3 pathway. Apoptotic cells were increased in sympathetic nerve inhibited rats and were decreased in parasympathetic nerve inhibited rats oppositely. It is concluded that the ANS could regulate the TNF-α expression and then lead to activate Caspase-3 in the liver apoptosis process. |
