臺灣博碩士論文加值系統

結核病(tuberculosis)是由結核菌群(Mycobacterium tuberculosis complex, MTBC)所引起，根據全球衛生組織(WHO)統計全球每年大約有900萬左右結核病新病例，造成100多萬人死亡，且近年來結核菌群的抗藥性日益增加。其中多重抗藥性(multidrug-resistant)的狀況也日益嚴重。且在2009年WHO將完全抗藥性結核菌（totally drug resistant tuberculosis, TDR-TB）分離且定義出來，由以上種種在抗結核菌活性新合成物的研究是目前重要的方向。
本研究中利用H37Rv標準菌株作為初步藥物篩選的目標菌株，且利用Alamar blue 作為指示劑培養後利用 ELISA READER測量其吸光值跟對照組比較菌株的生長抑制比例。另一方面利用Vero cell當model 測試藥物的毒性是否對細胞生長有所影響。實驗過程利用目前臨床常用ofloxacin(OFX)以及isoniazid(INH) 為positive control。
實驗結果發現，新合成的藥物TCH-3135以及TCH-3135與INH的合成複合物WCH-4378對結核菌的生長抑制有效濃度分別為2.05μg/ml以及0.96 μg/ml，在細胞毒性測試方面皆高於20μg/ml尚未對細胞有毒殺效果。根據以上結果，我們將TCH-3135以及WCH-4378以及WCH-4378兩個新合成物拿來測試對單一抗藥性結核菌菌株的抑制活性，結果發現TCH-3135僅對Rifampin-resistant (RIFr) strain有抑制效果：而WCH-4378對streptomycin-resistant (STMr) strain and Rifampin-resistant (RIFr) strain.皆有抑制其活性的效果，根據以上種種實驗結果我們可以發現TCH-3135與WCH-4378兩者有發展的潛力。

Tuberculosis (Mtb) is a disease caused by bacterium called Mycobacterium tuberculosis. According to the World Health Organization (WHO) reports, there are about 9 million new cases with Mycobacterium tuberculosis every year which caused more than 100 million people death. In recent years, the drug- resistant tuberculosis cases are increasing severely, especially the multidrug-resistant tuberculosis (MDR-TB). There are new cases with total drug-resistance defined by WHO in 2009. These cases illustrate that the development and discoveries of new anti-TB drugs in the world are important issues.
The growth of Mycobacterium tuberculosis is slow and takes 20 hours for each replication. The progress of tuberculosis study always needs more time. Therefore, reconstruction of rapid drug screening method becomes very important. In this Study, H37Rv standard strain was used as the initial drug screening strain and experiments method were using Alamar blue dye as an indicator for measuring the absorbance to compare the drug sensitivity and growth inhibition ratio between the control group and counter group. In cell viability test, Vero cell were used to be the model for drug cytotoxicity. The experiments use clinical drug ofloxacin (OFX) and isoniazid (INH) as a positive control.
Experimental results show that the measuring time of Mtb growth inhibition ration calculated by absorbance method is shorter than traditional method. The newly synthetic drug, TCH-3135, has the ability of anti-Mycobacterium tuberculosis on MIC50 concentration 2.05 g/ml. In the cytotoxicity results, TCH-3135 has no cytotoxicity on cells even the concentration is higher than 20μg/ml. TCH-3135 combined to the clinical one-line drug isoniazid (INH) was renamed WCH-4378. Mycobacterium tuberculosis MIC50 of WCH-4378 was 0.96μg/ml. As also, WCH-4378 has no cytotoxicity on cell until the concentration is higher than 20μg/ml. According to those results, when TCH-3135 and WCH-4378 treated with drug-resistant tuberculosis strains, Streptomycin-resistant (STMr) strain and Rifampin-resistant (RIFr) strain can also be inhibited. According to all result, we can prospect that TCH-3135 and WCH-4378 have the potential to become anti-mycobacterium tuberculosis drug.

中文摘要 i
英文摘要 iii
致謝………………………………………………………………………v
表目錄 ….ix
圖片目錄 X
前言 1
肺結核 1
結核分枝桿菌 2
（一）形態學 2
（二 ) 菌體成分與作用構成 4
（三） 分子生物學 4
結核菌之流行病學 5
結核病治療 7
實驗室診斷 10
抗結核菌藥物 11
1. Rifampin (RIF) 11
2. Isoniazid (INH) 12
3. Ethambutol (EMB) 13
4. Streptomycin (STM) 14
5. Pyrazinamide (PZA) 15
研究目的及架構 17
材料方法 18
一.合成藥物來源及配製 18
來源 18
配製 18
二. 結核菌來源 19
三.液態培養基配製 20
四.Positive control : ofloxacin（OFX）配製 20
五.結核菌濁度測試 21
六. 抗結核菌活性測試 21
七. 細胞毒性測試 26
八. 抗結核菌活性 MIC50值計算 30
九. 細胞毒性測試 IC50計算 31
十. TCH-3135與衍生物WCH-4378活性比較…………………….32
十一. 抗結核菌具單一藥物抗藥性之活性測試 32
十二. 實驗耗材、儀器列表 37
結果 38
結核菌濁度測試 38
抗結核菌活性測試 39
具單一抗藥性結核菌活性測試 42
討論 43
未來與展望 47
參考文獻 48

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