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研究生:陳聖岱
研究生(外文):Sheng-Tai Chen
論文名稱:CYP2E1, GTM1, NAT2基因多型性及T細胞活化與肺結核藥物過敏反應之關係
論文名稱(外文):CYP2E1, GTM1, NAT2 polymorphisms and specific T cell activation involved in the anti-tuberculosis agents-induced hypersensitivity
指導教授:洪舜郁
指導教授(外文):Shuen-Iu Hung
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:藥理學研究所
學門:醫藥衛生學門
學類:藥學學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:67
中文關鍵詞:抗結核藥物過敏T細胞
外文關鍵詞:Antituberculosis drugsHypersensitivityT cellsCYP2E1GSTM1NAT2
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世界上三分之二的人口生活結核病的威脅下,其中有大約百分之九接受抗結核藥物治療的病人會發生不良反應。遺傳,性別,年齡,過去用藥的歷史,都是可能造成不良反應發生的因素,但目前還沒有明確證據顯示哪一個因素最為關鍵。Isoniazid (INH)是一種結核病的主要藥物,主要是經過Cytochrome P450 2E1(CYP2E1)、Glutathione S-transferase Mu 1(GSTM1)、N-acetyltransferase 2(NAT2)三種酵素代謝。先前有研究指出,CYP2E1、GSTM1、NAT2三種酵素的基因多型性與結核藥物引起的肝毒性有關。本研究從結核藥物過敏病患代謝酵素基因多型性方面探討與結核藥物過敏反應發生的相關性;此外,由於結核病患通常同時接受多種結核藥物的治療,導致難以判斷是其中何種藥物引起不良反應,本研究另一目標即為建立一T細胞體外培養系統,用於判斷何種藥物引起不良反應的產生。本實驗利用基因定序方法探討結核藥物代謝酵素的基因多型性是否與結核藥物不良反應的發生相關,結果在結核藥物過敏病人身上發現3個會造成胺基酸改變的NAT2單核酸多型性(RS1208、RS1799930、RS1799931)出現頻率較高(n=17);CYP2E1、GSTM1、NAT2三個基因啟動子上也有兩個單核酸多型性與結核藥物的不良反應相關(n=8)。本次實驗針對代謝酵素基因定序的樣本數不夠充足,所以對於結核藥物代謝酵素基因的多型性是否與結核藥物不良反應的發生有相關性,尚且無法驟下定論。另一方面,利用結核藥物過敏病人的周邊血液單核球細胞,觀察不同結核藥物對於該細胞的刺激及反應,結果發現利用結核藥物刺激結核藥物特異性T細胞,某些特定結核藥物的確使得結核藥物特異性T細胞產生更多腫瘤壞死因子(TNF-alpha)及發生細胞增生現象。以上結果證明了利用T細胞體外培養系統辨別何種結核藥物引起不良反應的可行性。在本次研究中無法從藥物基因體學角度找到理想的答案,似乎代謝酵素基因多型性與結核藥物過敏的發生並無直接關係。不過從實驗觀察到,過敏病患藥物特異性T細胞的確會對特定藥物產生反應,或許再深入研究便可發現更明確的機轉,利用這些發現造福人群,避免藥害反應的不幸發生。
There is two thirds of the global population living under the threat of tuberculosis (TB), and about nine percent of the patients receiving TB drugs may develop adverse drug reactions (ADRs). Gene, gender, age and past medication history are risk factors for TB drugs-induced adverse reactions, but there is no confident evidence showing which factor is the most crucial. Isoniazid (INH) is one of the first-line antituberculosis medication in prevention and treatment. Cytochrome P450 2E1 (CYP2E1), Glutathione S-transferase Mu 1 (GSTM1), and N-acetyltransferase 2 (NAT2) are thought to be involved in its metabolism. Previous studies suggested that the genetic polymorphisms of these three drug-metabolizing enzymes are associated with TB drug–induced hepatitis. The first aim of this study is to clarify whether that polymorphisms of the genes that encode these enzymes associates with the TB drugs-induced adverse reactions. Furthermore, since tuberculosis patients are always administrated with multiple TB drugs, it is hard to distinguish which TB drug caused these adverse reactions. The second aim of this study is to establish an in vitro T cell culture system to find out which TB drug causes the adverse drug reactions. In this study, DNA sequencing method was used to prove the relationship between genetic polymorphisms of TB drug-metabolizing enzymes and the adverse drug reactions. Three SNPs (RS1208、RS1799930、RS1799931) causing residue changes were found in higher frequencies in the TB drug-hypersensitive patients (n=17). In addition, there are two significant SNPs associated with TB drugs–induced ADRs in the promoter region of GSTM1 gene (n=8). Nevertheless, the sample size for the genetic association study is not sufficient, therefore there is no conclusive result of the relationship between the genetic polymorphisms of TB drugs-metabolizing enzymes and the incidence of TB drug adverse reactions. In addition, cytokine releasing assay was used to exam the activation of TB-drug specific T cells upon the stimulation of TB drugs. Certain TB drugs were able to stimulate the T cells of hypersensitive patients to proliferate and secrete more TNF-alpha. These results suggest that using an in vitro T cell culture system to identify the culprit drug of anti-TB-agents induced hypersensitivity is feasible. The underlying mechanism of anti-TB-agents induced hypersensitivity mechanism is unclear and awaits the further in-depth study.
摘要 I
ABSTRACT III
目次 VI
表次 VIII
圖次 IX
附錄 X
1導言 1
1-1肺結核病 1
1-2 Isoniazid(INH) 1
1-3 Rifampicin(RIF) 2
1-4 Pyrazinamide(PZA) 2
1-5 Ethambutol(EMB) 2
1-6藥物不良反應 3
1-7藥物過敏 3
1-8結核藥物不良反應造成的問題 4
1-9藥物基因體學 5
1-10藥物代謝酵素的基因多型性與結核藥物不良反應 5
1-11藥物過敏與免疫反應 6
1-12研究目標 7
2材料與方法 8
2-1捐助者的血液 8
2-2 DNA提取 8
2-3 CYP2E1,NAT2,和GSTM1基因型 9
2-4 HLA Typing with Sequence-Specific Oligonucleotide Primed PCR (PCR-SSO) 9
2-5培養基和化學品 10
2-6 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) B細胞毒性測定法 10
2-7結核藥物特異性T細胞的培養 11
2-8酵素免疫分析法 11
2-9 RNA的抽取 12
2-10 cDNA (Complementary DNA)合成 12
2-11 Quantitative real time PCR (Q-PCR) 12
2-12結核病藥物特異性T細胞細胞類型 13
3實驗結果 14
3-1結核藥物過敏病人的臨床症狀 14
3-2 CYP2E1,NAT2,和GSTM1編碼區基因型 14
3-3 CYP2E1,NAT2,和GSTM1啟動子基因型 14
3-4結核藥物過敏病人HLA基因型 15
3-5 B細胞毒性試驗 16
3-6結核藥物刺激特異性T細胞增殖 16
3-7結核病藥物特異性T細胞帶有CD3+CD4+表面抗原 17
3-8特定結核藥物刺激周邊血液單核球細胞分泌TNF-alpha 17
3-9結核病藥物特異性T細胞培養上清液TNF-alpha含量 17
4討論 19
4-1 CYP2E1、GSTM1、NAT2基因多型性與抗結核藥物不良反應的關聯 19
4-2 不同SNP導致CYP2E1,NAT2,和GSTM1三個基因胺基酸改變代表的意義 19
4-3 GSTM1啟動子-1971及-1124兩個不同SNP所代表的意義 20
4-4結核病藥物特異性T細胞表面抗原類型所代表的意義 21
4-5結核病藥物特異性T細胞受到刺激後分泌TNF-alpha未如預期明顯 21
4-6以流式細胞儀觀察結核藥物過敏病人周邊血液單核球細胞經不同結核藥物刺激後TNF-alpha的分泌 22
5結論 23
6參考文獻 24
7表 28
8圖 43
9附錄 52
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