- 學位論文
棕櫚酸誘導HepG2肝細胞脂肪堆積與其差異性表現基因
The differential expressed genes in palmitic acid-induced lipid accumulation in HepG2
摘要
脂肪肝是中性脂肪堆積在肝細胞內的一種現象，其脂肪油滴主要以三酸甘油酯方式存在，其中非因酒精過量攝取所造成的脂肪肝稱之為非酒精性脂肪肝（non-alcoholic fatty liver disease，NAFLD）。因為NAFLD與代謝症候群具有密切的關聯，而且在造成慢性肝病的發生原因中愈顯常見，尤其NAFLD的發生是廣泛且普遍的，使得其重要性令醫學界愈加重視。學者通常藉由觀察脂肪肝發生時所產生的改變來了解NAFLD的病程原因。雖然已經有許多關於脂肪肝發生所改變的基因被報導，然而這通常是根據總體肝臟組織的分析數據所得結論，但是對於肝細胞在NAFLD歷程的重要性，則很難釐清。為了解構肝細胞在NAFLD中所扮演的角色，本研究以棕櫚酸作為誘導物，利用200 ?嵱棕櫚酸處理人類肝癌細胞株HepG2，可在第二天形成脂肪肝的細胞模式。經由基因表現晶片分析，發現在第二天及第六天表現的差異基因多涉及細胞凋亡、發炎反應調控、脂類代謝、細胞骨架與分子轉移調控，其中進一步以RT-PCR確認lymphocyte antigen 96（LY96）、glycerol kinase、interleukin-8在一至四天內因棕櫚酸而增加基因表現。利用富含20%豬油飼料餵食C57BL/6JNarl小鼠在第16.5週產生脂肪肝現象，其肝臟LY96的表現量確實增加。在LPS的例子中，LY96中介NFkB的活化途徑，不過根據我們的發現，棕櫚酸會增加LY96的表現，但是卻會抑制NF?羠的活性。推測LY96的增加可能具有其他生理意義，後續將朝向抑制內生性LY96的表現以探究其在肝細胞脂肪堆積的過程中所扮演的角色。
並列摘要
Non-alcoholic fatty liver disease (NAFLD) is defined histologically by the accumulation of lipid droplet, mostly in the form of triglyceride, in hepatocytes. Recently, NAFLD becomes a common cause of chronic liver disease. Especially, NAFLD is cross-linked with metabolic syndrome. The prevalence of NAFLD in Taiwan is estimated to be one-third in general adult population. In order to study the pathogenesis of NAFLD in hepatocyte level, we induced human hepatoma cell line HepG2 into steatotic state with 200 ?嵱 palmitic acid for 2 to 6 days. Subsequentially, we surveyed the RNA expression profile with microarray gene chips. The differential expressed genes at day 2 and day 6 were clustered into 10 functional groups, including cell cycle regulation, anti-apoptotic regulation, inflammatory response, carbohydrate and lipid metabolism, cytoskeleton and intracellular trafficking, muscle contraction and cysteine protease inhibitors. The up-regulation of lymphocyte antigen 96 (LY96), glycerol kinase, interleukin-8 were validated with RT-PCR. In high fat-diet induced NAFLD mouse model, we observed the increase of LY96 RNA expression in the mouse liver. Because palmitate treatment increased LY96 expression but inhibit NF?羠 activity, it suggested that LY96 may play unknown roles during lipid influx. We shall silence the endogenous LY96 to study the roles of LY96 in the development of steatosis.