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研究生:蕭愛琪
研究生(外文):Ivy Hsiao
論文名稱:尼古丁對人成骨細胞組織毒性反應之機轉
論文名稱(外文):Mechanisms of apoptosis in bone cells after nicotine induction
指導教授:洪志遠洪志遠引用關係王若松王若松引用關係
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:臨床牙醫學研究所
學門:醫藥衛生學門
學類:牙醫學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:英文
論文頁數:82
中文關鍵詞:尼古丁人成骨細胞毒性反應
外文關鍵詞:NicotineBone cellsApoptosis
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中文摘要
尼古丁是一種自香煙萃取出來的主要成份,被廣泛的認為對人體有致癌的作用,人體內成骨細胞組織為構成骨生成最重要的一種細胞,當骨整合人工植牙手術後,成骨細胞會分裂,分化並愈合,使骨細胞組織與人工牙根植體接合成為一體。
然而影響植體周圍骨質不良與相鄰骨組織的骨整合不理想之外在因素中以尼古丁對人體成骨細胞所造成的毒性反應被指是主要的因素。各家學者皆有不同的看法,根據不同的學者,應用不同的研究方法,作出來的結果也不一樣。為了瞭解尼古丁對人體成骨細胞所造成的毒性反應,我們選用二種成骨細胞株做實驗,分別是 MG63 (mutant p53) 和U2OS (wild type p53),在固定的時間點蒐集尼古丁處理後之細胞,使用流式細胞儀分析發現,高量的尼古丁處理四十八小時之後造成MG63及U2OS停滯在sub-G1階段。之後我們選用DNA Fragmentation Assay之方法來評估這些細胞在高濃度的尼古丁處理下是否會造成細胞DNA的斷裂,然而這兩株細胞的結果皆為陽性反應。但是MG63這株細胞在TUNEL染色下,呈現陽性反應,表示細胞凋零現象之進行,然而U2OS這株細胞並沒有呈現陽性反應。因此我們希望進一步利用西方點墨法來確定p53與細胞週期停滯及細胞凋零之相關性,最後再分析p53下游基因Bax, Bcl-2, p21之表現。結果顯示在MG63之細胞株中,尼古丁的處理使得parp蛋白質表現增強 U2OS之細胞株中,8mM的尼古丁處理使得p53以及其下游的基因p21蛋白質表現增強。顯示尼古丁引起的MG63細胞凋零現象並不是經由p53之途徑,然而尼古丁引起的MG63細胞凋零現象並不是經由p53之途徑,
然而尼古丁引起的U2OS細胞死亡現象是經由p21之相關途徑而造成的。
由此可見,尼古丁對人體成骨細胞組織作用之機轉有兩大類,一為急速死亡,另一為自然凋亡,急速死亡現象常見於高濃度化學物質作用於細胞,細胞急速崩解,釋放大量炎性化學物質。別於急速死亡,自然凋亡乃當細胞受外力傷害後,在無法修復正常生理機能的情況下,所進行之細胞萎縮現象。
Abstract
Nicotine is a major component of cigarette smoke and has been postulated to play an important role in malignancy. Smoking and nicotine have been shown to delay the revascularization and incorporation of bone grafts.
This study looks at the mechanism of apoptosis in bone cells after nicotine induction. For this study 2 cell types have been selected : MG63 human osteosarcoma cell (p53 mutant type) and U2OS human osteosarcoma cell (p53 wild type). The detection of apoptosis first involves (a) Cytotoxic assay : a colorimetric method for determining the number of viable cells in proliferation.The MTS tetrazolium compound is bioreduced by cells into a colored formazan product and assays can be read by recording the absorbance at 490nm (b) Flow cytometry : Cell cycle analysis of MG63 and U2OS cells responding to nicotine at different concentrations and time intervals. (c) DNA fragmentation : The DNA of apoptotic cells generally migrates as a ladder of 180-200bp multimers on agarose gel. (d) TUNEL (TdT-mediated dUTP Nick End Labeling ) : Measures the fragmented DNA of apoptotic cell by the incorporation of fluorescein -12-dUTP at 3’-OH DNA ends.These can then either be visualized directly by fluorescence microscopy or quantitated by flow cytometry..(e) Western blot analysis of (a) MG63 : Assay for p53 independent pathways (2) U2OS : Assay p53 gene and its down stream genes expression. MG63 and U2OS are treated with nicotine at different concentrations (C, 4mM, 8mM, 16mM) and collected at time intervals of 24 and 48 hours.
Therefore, MG63 after induction of nicotine shows a dose dependent relationship in causing apoptosis. U2OS after the induction of nicotine does not show clear evidence of apoptosis but rather cell cycle arrest. The data suggested that the induction of nicotine on bone cells may induce apoptosis in MG63 and cause cell cycle arrest in U2OS cells.
Table of contents
Abstract……………………………………………..1
List of Figures……………………………………...5
Background
I. Dental implants and osseointegration…6
II. Nicotine and its adverse effects………..8
III. Apoptosis………………………………..25
.
Specific Aims………………………………….…. 44
Materials and Methods………………………… .45
Results………………………………………….…51
Discussion……………………….…………….….68
References……………………….……………….73
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