篩選及分析斑蝥類化合物對於抑制人類肝癌細胞株和B型肝炎病毒複製之影響; Screening and Characterization of Cantharidin analogues That Suppress Hepatitis B Virus Replication in Human Hepatoma cell line

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Title:	篩選及分析斑蝥類化合物對於抑制人類肝癌細胞株和B型肝炎病毒複製之影響;Screening and Characterization of Cantharidin analogues That Suppress Hepatitis B Virus Replication in Human Hepatoma cell line
Authors:	王舒芳;Shu-Fang Wang
Contributors:	中國醫藥學院醫學研究所
Keywords:	斑蝥類化合物;2.2.15細胞株;人類肝癌細胞株;B型肝炎病毒;cantharidin analogues;2.2.15 cell line;human hepatoma cell line;HBV
Date:	1999
Issue Date:	2009-12-02 14:11:23 (UTC+8)
Abstract:	B型肝炎為亞非地區廣泛流行之疾病，尤其在台灣地區有相當高比例帶原者。由流行病學研究顯示，B型肝炎帶原者罹患肝癌比例遠高於正常人，並且發現大部分肝癌細胞中都含有B型肝炎病毒 (HBV) DNA，由此可知B型肝炎病毒感染和肝細胞癌化有密切的關係，我們試著從傳統中藥斑蝥探討對肝癌細胞株和B型肝炎病毒複製有影響之活性成分。斑蝥素 (cantharidin)為斑蝥的主要有效成分，已被證實具有抗腫瘤作用，還會引起白血球增加及出血等症狀。目前仍然未知其藥理作用機轉，因此我們計畫評估斑蝥類化合物對於肝癌細胞株及B型肝炎病毒的影響。斑蝥類化合物對肝癌細胞株2.2.15細胞的毒殺性，其CC50值 (影響50%細胞數目的藥物濃度)分別為Na CTD (disodium cantharidate)＝ 3.69mM，Na DHNCTD (disodium dehydronorcantharidate)>50mM，NCTD (norcantharidin)＝14.12mM，DHNCTD (dehydronorcantharidin)＝16.61mM。藉由Wright-Giemsa stain染色法，發現2.2.15細胞在加入12.5mM Na CTD培養12天後，出現空泡化、型態改變。經由流式細胞分析儀研究2.2.15細胞在斑蝥類藥物作用後DNA分佈情形，發現在高濃度的Na CTD及NCTD作用下會使細胞週期的S期受到抑制。斑蝥類化合物對於HBV病毒的影響，藉由分析細胞外的HBsAg的含量來檢測藥物抗病毒的作用。應用EIA套裝試劑測量PLC/PRF/5細胞培養液中HBsAg的濃度，細胞數目以錐藍排除法來計數，分別考慮其CC50 (影響50% PLC/PRF/5細胞數目的藥物濃度)及IC50 (影響50% HBsAg分泌量的藥物濃度)，所計算出的SI值 (selective index＝CC50/ IC50)為NCTD>Na DHNCTD>DHNCTD>Na CTD依序遞減。根據實驗結果顯示，隨著藥物濃度上升，細胞外的HBsAg濃度呈劑量依賴性減少。在本篇研究中，我們討論斑蝥類化合物對於肝癌細胞株及抗病毒作用的影響，由本實驗的初步數據，這些化合物將可被考慮用來發展為抗肝癌及抗B型肝炎病毒的藥物。; Chronic hepatitis B virus is one of the most widespread disease in Asia and Africa. Especially in Taiwan, there is having prevalence of hepatitis B carriers. The epidemiological studies showed that the indent rate of hepatoma in those carriers is far beyond the normal population. Another finding showed that a majority of the hepatoma cell line contains hepatitis B virus DNA, hence a high correlation in between them is expected. We have tried to trace the affective components of hepatoma cell line and hepatitis B virus in Mylabris. Cantharidin, the active constituent of mylabris, has been demonstrated to possess antitumor properties, and causes leukocytosis and hemorrhagic property. The pharmacological mechanism is still unknown. Our purpose is to evaluate the effect of cantharidin analogues on hepatoma cell line and hepatitis B virus. The drug concentration caused 50% cellular cytotoxicity in 2.2.15 cell number (CC50) of cantharidin analogues, Na CTD (disodium cantharidate) =3.69mM, Na DHNCTD (disodium dehydronorcantharidate)>50mM, NCTD (norcantharidin) =14.12mM, DHNCTD (dehydronorcantharidin) =16.61mM, respectively. By Wright-Giemsa stain, the morphology in 12.5mM Na CTD for 12days can observed vacuolization and degeneration changes of the 2.2.15 cell line. By the Fluorescene- activated cell sorting (FACS) analysis of DNA profiles in 2.2.15 cell line, the distribution of cell cycle phase induced that high dose of Na CTD and NCTD inhibited the S phase. The antiviral effects of cantharidin analogues were measured by analysis of HBsAg contents. The HBsAg of PLC/PRF/5 cells in culture medium were measure by enzyme immunoassay (EIA) kits. Cell numbers were determined by trypan blue exclusion. The selective index (SI= CC50/ IC50) sequentially decreased from NCTD>Na DHNCTD>DHNCTD>Na CTD. The experiment revealed that the amounts of HBsAg were decreased by a dose-dependent manner for each drug. In this study, we describe the effect of hepatoma cell line and antiviral activity of cantharidin analogues. These compounds should be considered for development as anti-cancer and anti-HBV drugs.
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