核苷;誘導人類肝癌細胞株HepG2與Hep3B細胞程式凋亡之影響

Effects of nucleosides on the induction of apoptosis in HepG2 and Hep3B cells.

邱哲琳(Che-Lin Chiu)

指導教授 ： 楊素卿博士

臺北醫學大學/公共衛生暨營養學院/保健營養學研究所/碩士(2003年)

摘要

本研究針對核苷誘導人類肝癌細胞株HepG2和Hep3B 細胞程式凋亡之影響進行探討。實驗以不添加(控制組)或添加核苷之培養液進行HepG2和Hep3B之培養。本實驗使用六種核苷包括：Inosine(I)、Cytidine(C)、Uridine(U)、Thymidine(T)、Adenosine(A)和Guanosine(G)。在37℃、5%CO2環境下培養，3、6、12、18和24小時後，收集細胞內、外液進行分析。由細胞存活率結果可知，由HepG2方面，於培養液中添加30mM I、30mM C、30mM U、30mM T、0.5mM A和1mM G，於培養後12和24小時，會造成細胞存活率顯著較控制組低(p＜0.05)；而Hep3B方面，則是添加30mM I、30mM C、30mM U、30mM T後，培養12和24小時，造成細胞存活率顯著較控制組低(p＜0.05)，0.1mM A和0.2mM G則無差異。細胞程式凋亡現象觀察之結果，在HepG2細胞週期第12小時，C、U、T、A與G組別皆可使sub G0期顯著較控制組高(p＜0.05)，而Hep3B在細胞週期第24小時，C、U、T與A組別皆可使sub G0期顯著較控制組高(p＜0.05)。Caspase-3活性結果方面，HepG2在第6小時只有T組顯著較控制組高(p＜0.05)，而Hep3B在第6小時，I、T、A與G組皆可使Caspase-3活性顯著高於控制組(p＜0.05)。P53與p21蛋白質在HepG2中皆可測得，但在Hep3B中皆無法測得。細胞程式凋亡作用機轉結果，由細胞外液水溶性Fas-Ligand結果可知，HepG2在第18小時發現，C、U、T與A組別皆顯著較控制組高(p＜0.05)，而Hep3B在第12小時，I、T、A和G組皆可使水溶性Fas-Ligand濃度顯著較控制組高。另外，由細胞外液TNF-α結果與細胞內cytochrome c蛋白質結果可知，在HepG2與Hep3B細胞中皆無法測得TNF-α與cytochrome c蛋白質。由本研究結果可知，嘌呤核苷與嘧啶核苷雖然可以顯著降低人類肝癌細胞株HepG2與Hep3B之細胞存活率，但是由細胞程式凋亡現象及作用機轉之結果，無法證實其細胞存活率降低是由於促進細胞程式凋亡所引起。 關鍵字：HepG2、Hep3B、細胞程式凋亡、核苷

關鍵字

HepG2 ； Hep3B ； 細胞程式凋亡 ； 核苷

並列摘要

The purpose of this study is to investigate whether nucleosides can induce the apoptosis in human hepatoma HepG2 and Hep3B cells or not. The nucleosides which were used in this study included inosine (I), cytidine(C), uridine (U), thymidine (T), adenosine(A) and guanosine(G). Cells were incubated by the mediums with or without nucleosides at 37℃ in a 5% CO2 humidified atmosphere. Then, the cells and mediums were collected and analyzed after incubation. It was found that the cell viability was significantly decreased in HepG2 cells which were treated with 30mM I, 30mM C, 30mM U, 30mM T, 0.5mM A and 1mM G after 12 and 24 hours incubation (p＜0.05). On the other hand, the reduction of the cell viability in Hep3B which were incubated with 30mM I、30mM C、30mM U and 30mM T after 12 and 24 hours were also observed(p＜0.05). But the cell viability in Hep3B, which was incubated with 0.1mM A and 0.2mM G after 12 and 24 hours, were no different. About the apoptotic phenomenon, the cell percentages of Sub G0 phase were significantly increased in HepG2 cells, which were treated with nucleosides such as C, U, T, A and G (p＜0.05). And, the Hep3B cells in sub Go phase were also raised, when the medium were added with C, U, T and A. Furthermore, the caspase-3 activity was increased when HepG2 cells were incubated with T (p＜0.05). In the Hep3B cells, the caspase-3 activities were increased when the mediums contained I, T, A and G (p＜0.05). The expressions of p53 and p21 were detected in HepG2 cells. However, there was no expression of p53 and p21 in Hep3B cells. To investigate the mechanism of apoptosis induced by nucleosides, it was found that the content of soluble Fas ligand (sFas L) was increased in HepG2 cells following C, U, T and A treatment (p＜0.05). Moreover, the Hep3B cells was also increased in sFas L contents when mediums contained I, T, A and G (p＜0.05). But, there was no detection of TNF-α and cytochrome C in HepG2 and Hep3B cells. In conclusion, although purine and pyrimidine nucleosides could reduce the cell viability, that could not confirm reduction of the cell viability by apoptosis in results of apoptotic phenomenon and mechanism of apoptosis. Key words : HepG2、Hep3B、apoptosis、nucleosides

並列關鍵字

HepG2 ； Hep3B ； apoptosis ； nucleosides

參考文獻

Adams JM and Cory S (1998) The Bcl-2protein family: arbiters of cell survival. Science 281:1322-1326

Araragi S, Kondon M, Kawase M, Satio S, Higashimoto and Sato M. (2003) Mercuric chloride induces apoptosis via a mitichondrial-dependent pathway in human leukemia cells. Toxicology 184: 1-9

Ashkenazi A and Dixit VM (1998) Death receptors : singaling and modulation. Science 281: 1305-1308

Batiuk TD, Schnizlein-Bick C, Plotkin Z and Dagher PC (2001) Guanine nucleosides and jurkat cell death: roles of ATP depletion and accumulation of deoxyribonucleotides. Am J Physiol Cell Physiol 281: 1776-1784

Buren CTV and Rudolph F (1997) Dietary nucleotides: a conditional requirement. Nutrition 13: 470-472