C 型肝炎病毒非結構蛋白NS5A誘導Huh-7肝癌細胞發生凋亡之研究

A study on the molecular mechanisms involved in HCV NS5A-induced apoptosis of Huh-7 cells

洪靖雯(Jung-Uen Hong)

指導教授 ： 鄭鈞文 ； 邱慧玲

中山醫學大學/醫學院/生化暨生物科技研究所/碩士(2008年)

https://doi.org/10.6834/CSMU.2008.00026

摘要

C型肝炎病毒感染可導致嚴重的肝臟疾病，長期的慢性感染甚至會演變成肝硬化或肝癌。當宿主細胞受到C型肝炎病毒感染的時候，可藉由C型肝炎病毒本身外套膜蛋白的高度變異來躲避宿主免疫系統的攻擊；另外一種方式則是透過病毒蛋白的作用來抑制宿主免疫系統的產生或是促使細胞凋亡來幫助病毒繁殖擴散，導致組織產生持續性的發炎反應，進而引起慢性肝炎、肝硬化以及肝癌。C型肝炎病毒的非結構蛋白NS5A已經被證實是個多功能的蛋白，在細胞的調控以及病毒持續性感染與致病機轉有關的路徑均有參與，但NS5A是否參與急性肝炎的致病機制或病毒的繁殖擴散，卻鮮少被研究。因此藉由此研究來探討NS5A是否有導致細胞凋亡的能力，更進一步的探討此蛋白對於細胞凋亡相關分子的影響。經由MTT assay、DNA fragmentation以及western blot等分析方式，我們發現NS5A確實會導致Huh-7肝癌細胞產生細胞凋亡，並且促進procaspase 8、procaspase 9及procaspase 3的活化以及使得PARP蛋白受到切割，cytochrome c、Bax及tBid蛋白表現量也因而增加，同時Bid及Bcl-2蛋白表現量減少。根據上述結果可以發現，非結構蛋白NS5A會使細胞凋亡抑制蛋白Bcl-2的蛋白表現量減少，並且藉由透過切割活化procaspase 8，將Bid的C端切除而形成tBid蛋白，使tBid蛋白移動到粒線體細胞膜上使得Bax蛋白的活化，導致粒線體膜電位改變，而將cytochrome c釋放至細胞質當中，促使procaspase 9蛋白活化，並繼續活化下游的procaspase 3蛋白，並導致procaspase 3的受質PARP蛋白受到切割而進行細胞凋亡。

關鍵字

C行肝炎病毒 ； 非結構蛋白 ； 細胞凋亡

並列摘要

Hepatitis C virus (HCV) infection may lead to liver diseases of various severities, from acute hepatitis to hepatocellular carcinoma (HCC) with poor prognosis. Although the mechanism for viral persistence is poorly understood, the high mutation rates of viral envelope proteins and the suppression of the host immune system are believed to involve in chronic infection. HCV NS5A protein has been demonstrated to be a versatile protein involved in many cellular events and various pathways related to viral persistence or pathogenesis while the involvement of NS5A in apoptotic induction to lead to hepatic injury has been less investigated. Therefore, in this we studied NS5A may induce apoptosis of Huh-7 cells and then investigated the underlying mechanisms. The results showed that NS5A indeed could induce apoptosis of Huh-7 cells by promoting the activation of procaspase 8, procaspase 9 and procaspase 3 and cleaveage of PARP, while protein expression levels of cytochrome c, Bax and tBid were up-regulated in a time-dependent fashion, while Bid and Bcl-2 protein were down-regulated in a same manner. In summary, NS5A may induce apoptosis of Huh-7 cells through a mitochondria-dependent pathway.

並列關鍵字

HCV ； NS5A ； apoptosis

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