探討在檳榔鹼所誘導的染色體結構異常中氧化壓力所扮演的角色

中山醫學大學機構典藏 CSMUIR > 醫學科技學院 > 生物醫學科學學系暨碩士班 > 博碩士論文 > Item 310902500/12223

Please use this identifier to cite or link to this item: https://ir.csmu.edu.tw:8080/ir/handle/310902500/12223

Title:	探討在檳榔鹼所誘導的染色體結構異常中氧化壓力所扮演的角色 The role of ROS in the context of chromosome aberrations induced by arecoline.
Authors:	黃左丞 Hung, Zuo-Cheng
Contributors:	中山醫學大學:生物醫學科學學系碩士班;李月君
Keywords:	檳榔鹼;染色體結構異常;活性氧物種;遺傳毒性 Arecoline;chromosome aberration;ROS;Genotoxicity
Date:	2015
Issue Date:	2015-09-21T03:02:10Z (UTC)
Abstract:	在許多的研究中已經證實檳榔成分中一個主要的生物鹼－檳榔鹼(arecoline)，會對不同種類的細胞造成細胞毒性。及在小鼠的細胞中造成染色體結構異常，但目前沒有人類細胞的相關研究。不過也有研究發現檳榔鹼能誘導多種人類細胞的氧化壓力上升。而氧化壓力已廣泛地被知曉涉及細胞毒性、細胞週期損害、細胞功能損害及DNA傷害。故本研究便想要探討在檳榔鹼所誘導的氧化壓力在染色體結構異常中所扮演的角色。本研究對於檳榔鹼所造成的細胞生長、毒性、氧化壓力的變化、細胞凋亡的產生及染色體結構異常，分別以MTT分析法、DCFH-DA染色法、8-OHdG螢光標定、Annexin-V/PI雙染色試驗及染色體結構異常(CA)分析來進行測量。在本研究結果中，顯示中國倉鼠卵巢細胞(CHO-K1)及人類牙齦上皮細胞(S-G)在經過檳榔鹼的處理後，皆會產生細胞毒性，並且比起S-G，CHO-K1對於檳榔鹼有較高的耐受力。在CA分析的結果顯示，檳榔鹼在兩種細胞中皆會產生染色體結構異常，並隨著檳榔鹼濃度提升而增加；更進一步的，CA與處理時間的趨勢結果顯示當檳榔鹼濃度較低時兩種細胞所被誘導的染色體結構異常並不隨時間增加，而當濃度與時間增加時，則出現第二階段與第三階段的CA增幅。在活性氧物質(ROS)的測定結果顯示出，檳榔鹼會在CHO-K1細胞中造成氧化壓力上升，然而在S-G細胞中則沒有測得；進一步也確認了檳榔鹼所誘導的氧化壓力在CHO-K1中會形成8-OHdG，進而可能造成DNA的損傷。在細胞同時處理檳榔鹼及GSH的研究中更進一步的顯示了，GSH能有效的抑制檳榔鹼在CHO-K1細胞中所誘導的ROS及8-OHdG的產生且降低染色體結構異常率；然而，不被檳榔鹼誘導出氧化壓力的S-G細胞卻也能藉由GSH抑制檳榔鹼所造成的染色體結構異常。在CA與處理時間的趨勢中也顯示出當CA程度超過一個閾值後GSH才開始有效降低CA，並將其降回基準線。然而，由於GSH已知在細胞內參與多種不同功能，並其對活性氧物質(ROS)的清除效果不止於單一種ROS，所以我們更進一步分別以SOD(超氧化物岐化酶)、甘露醇及catalase(過氧化氫酶)來測定超氧陰離子(O2￣)、氫氧自由基(˙OH)及過氧化氫(H2O2)所造成的氧化壓力。在DCFH-DA檢測的結果顯示，過氧化氫酶(CAT)能有效抑制檳榔鹼在CHO-K1所誘導的氧化壓力，故此氧化壓力可能為過氧化氫(H2O2)所造成的。在細胞共同處理了檳榔鹼與過氧化氫酶(CAT)的測試結果也顯示，過氧化氫酶(CAT)僅能在CHO-K1細胞中抑制檳榔鹼所誘導產生的染色體結構異常，而且抑制的效果並不如GSH有效。綜合CA與處理時間的趨勢，我們推測檳榔鹼本身便會直接造成CA，但在高濃度或更長的檳榔鹼處理時間中ROS會被誘導出現，可能造成第二階段與ROS傷害相關的CA，當處理濃度和時間都持續增加時，可能造成第三階段與細胞凋亡相關的CA，在此研究結果中也發現GSH與CAT不同，GSH不只降低了會被誘導出ROS的CHO-K1的CA，也降低了不會被誘導出ROS的S-G的CA，這樣的結果顯示出了GSH在對染色體結構異常的調控上也扮演了非僅只於ROS清除劑的角色。 Arecoline, one of the major alkaloid in Areca nut. It has been evidenced arecoline-induced cytotoxicity in various mouse and human cell types, while the induced chromosome aberrations only in mouse cell lines but not in human cell lines. The arecoline-induced ROS (reactive oxygen species) was also identified in several human cell lines. It was known that the oxidative stress from the elevated ROS impairs cellular functions involving in cytotoxicity, cell cycle arrest, and DNA damage, etc. In this study, we identified identify the role of ROS in the context of the arecoline-induced chromosome aberrations. We used the in vitro chromosome aberration assay to evaluate the extent of chromosome aberrations in the condition with arecoline-induced ROS or scavenged ROS. The cell growth and cytotoxicity were assessed using MTT assay and PI staining. Apoptosis was analyzed using the Annexin-V/PI assay. The ROS was quantitated using DCFH-DA detector and the oxidative. And the ROS induced oxidative DNA damage was detected by immunofluorescence using 8-OHdG antibody. The result of cytotoxicity and chromosome aberrations showed that CHO-K1(Chinese hamster ovary cell line) cells were more resistant to arecoline than S-G (human oral gingival epithelial cell line) cells. The dose of arecoline induced chromosome aberrations was higher in S-G cells than CHO-K1 cells. Moreover, the kinetics of CAs-time showed that the arecoline induced CAs would reach a steady state at low dose treatment in CHO-K1 and S-G cells. At higher dose for long term treatment, it would appear second and third phase and tremendous CAs. Surprisingly, we found that arecoline induced cellular ROS in CHO-K1 cell, but undetectable ROS in S-G cell. The result of 8-OHdG immunofluorescence showed this induced ROS cause the oxidative DNA damage. Furthermore, co-treated with arecoline and GSH (a ROS scavenger) in CHO-K1 and S-G cells, the ROS and 8-OHdG signal induced by arecoline in CHO-K1 cell can be scavenged and the chromosome aberrations decreased in both CHO-K1 and S-G cells. Interestingly, the kinetics of CAs-time showed that the GSH started to inhibit CAs when CAs reached a threshold and this inhibition was not complete but to baseline in both CHO-K1 and S-G cells. We found that SOD and mannitol did not decrease the level of ROS induced by arecoline, but catalase reduces the level of ROS induced by arecoline in CHO-K1 cell. Furthermore, co-treated with arecoline and catalase in CHO-K1 and S-G cells, the chromosome aberrations also decreased in CHO-K1 but not in S-G cells. Moreover, catalase has less effective than GSH on decreasing CAs. Based on the kinetics of CAs-time, we suggested that arecoline itself directly cause dose-dependent CAs. At higher dose or long term treatment, the second phase of CAs was resulted from the induced ROS and the third phase and tremendous CAs were proposed from the apoptotic effect. The GSH decreased the CAs in both ROS-producing CHO-K1 cells and ROS-nonproducing S-G cells. This suggested that the GSH might have other different effect on chromosome aberrations in addition to as a ROS scavenger.
URI:	https://ir.csmu.edu.tw:8080/ir/handle/310902500/12223
Appears in Collections:	[生物醫學科學學系暨碩士班] 博碩士論文

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