体外试验多巴胺对肝细胞生存能力之影响.DOC

PAGE 2 體外試驗多巴胺對肝細胞生存能力之影響 Impact of Dopamine on Viability of Hepatocytes in vitro 研究生：陳堃焯 Chen, Kun-Cho 指導教授：孫灼均、高英賢、林貞信、吳明昌 Cheuk-Kwan Sun , Ying-Hsien Kao , Jen-shinn Lin , Ming-Chang Wu 【摘要】 多巴胺是屬於兒茶酚胺類的一種神經傳導物質。在臨床上，多巴胺可用來維持腦死病患血液動力學上的穩定，促進器官的捐贈應需要適當的灌注以維持其功能，例如肝臟、心臟和腎臟。然而多巴胺對於移植後的肝臟細胞是否產生負面效應仍具爭議。本研究目的是確定多巴胺對於肝細胞之生物效應。正常大鼠肝臟細胞株 (clone-9 cell) 在正常細胞培養模式下經由104 ng/mL多巴胺處理72小時後，細胞數目有減少的情形且細胞存活率呈現下降趨勢 (p 0.01)。細胞週期方面，分別以0~104 ng/mL多巴胺處理三天後，細胞週期停滯於G1期但未發現細胞有發生凋亡現象。RT-PCR顯示經多巴胺長時間處理下 bcl-2、hsp70 與 bax基因之比率皆有顯著性下降 (p 0.01)。西方墨點法證實細胞內NF-κB p65蛋白在高劑量多巴胺作用後，表現量明顯下降 (p 0.01)，且 Hsp70/Bax 和 Bcl-2/Bax 比值也呈下降趨勢 (p 0.01)。多巴胺處理後的培養細胞再利用添加 lipopolysaccharide (LPS) 模式進行觀察其可能對NF-κB依存性訊息傳遞 (NF-κB-dependent signaling) 的影響，結果發現多巴胺的刺激會抑制經由LPS 所誘發的I-κB磷酸化，進而導致NF-κB p65無法入核 (nuclear translocation)。這些實驗結果支持多巴胺可能藉由調控 NF-κB p65 表現量、I-κB 磷酸化及 NF-κB 訊息傳遞活性來減少Bcl-2等抗凋亡蛋白的表現，最後可能促使細胞走向凋亡。綜合上述結果高劑量之多巴胺可能對於正常肝臟細胞與肝臟摘取後之肝功能有不良之影響。 關鍵字: 多巴胺、肝臟細胞、細胞凋亡、細胞週期、肝臟摘取 【Abstract】 Dopamine (DA) is a neurotransmitter structurally similar to catecholamines. DA has been clinically used to maintain hemodynamic stability of brain dead patients, thereby facilitating donation of organs that require adequate perfusion for proper function such as the liver, heart, and kidneys. However, it is still controversial whether DA treatment deteriorates physiological functions of the grafts transplanted. This study aimed at determining the biological effect of DA on cultured clone-9 hepatocytes. DA treatment at 104 ng/mL under normal culture conditions for 72h significantly reduced hepatocyte numbers as well as cell viability (p 0.01). Flow cytometrical analysis revealed G1 growth arrest but no appearance of apoptotic sub-G1 subpopulation. Long-term exposure to high dose of DA decreased both ratios of Bcl-2/Bax and Hsp70/Bax at transcriptional and translational levels (p 0.01), as revealed by RT-PCR and Western blotting, respectively. Additionally, intracellular content of NF-κB p65 was prominently reduced. The b