結核病(Tuberculosis, TB）是常見並可致命的傳染病，文獻中提及全球人口約有1/3感染此結核分枝桿菌。疑似結核分枝桿菌感染病人，確診依據須符合1)臨床症狀 2)放射學影像 3)細菌培養結果。而細菌培養結果需長達1-2個月才能見分曉。確診後病人是否能確實配合用藥，針對治療療效監控，應藉由檢驗方法協助佐證。
就目前結核菌檢驗而言，抗酸性細菌染色法（Acid fast stain, AFs），只需30-60分簡便快速，但缺點為檢出率低，仍以細菌培養結果為黃金標準。文獻指出，被感染結核分枝桿菌個體，會刺激反應輔助T细胞（T helper cell）表現，引發Th1和Th17產生不同細胞激素（Cytokine）及Treg細胞內免疫反應，以對抗外來結核分枝桿菌感染。本研究期望能及時利用結核分枝桿菌病人血清所激發不同細胞激素含量變化，能有效區別TB和非TB感染和用藥前後之Cytokine變化相關性，進而輔助臨床診斷之治療過程監控。
本研究共收集44個檢體(34組病人)，包含確認19個TB感染(治療前)及配對10個治療後(6個月完整療程結束)，潛伏性肺結核陽姓病人(5人)，及確認為肺結核陰性之對照組病人(10人)，以此四組分析Th1，Th2，Th17及Treg相關CytokineIL-1β, IL6, IL4, IL12, IL17A, IFN-γ, TNF-α, TGF-β1分泌量關係。實驗結果在TB感染病人群組中與健康人比較，前者於IL6, IL17A, IFN-γ都有較高比率呈現且呈獻統計上顯著之差異；而相對於治療6個月後與健康人相比較，前者在IL12，IL17A有較高比率呈現，另TNF-α， TGF-β1則呈現下降比率，且這4個Cytokines在統計上皆呈顯著之差異；另外潛伏性TB病人與健康人比較，前者呈現 IL17A及 TGF-β1有較高比率呈現，但 IL6呈現下降比率。
從實驗中發現 IL17在不論於TB發病，治療後或潛伏TB病人組比上健康人皆有較高比率呈現及統計學上顯著之差異，因健康人群組測得之 IL17值幾乎為0，故推測IL17應可作為結核菌感染治療指標，且依據 IL17存在含量及下降量，亦可作為治療成效及觀察預後指標；另發現偵測TB發病病人群 IL6有較高比率呈現，可作為對照痰中有菌之指標，若治療後成效不佳痰中仍有菌時，IL6含量與治療前比較仍居高不降，可做為需更強化治療或是持續隔離之依據。故結論 IL17及 IL6在健康對照組中相對含量及比率皆低，故其測值上升可做為TB感染且是否為正值傳染性病人指標，給肺結核感染病人的診斷帶來新的曙光。

Tuberculosis (TB) is a common and lethal contiguous disease. Nearly one-third of the world's population has been infected by Mycobacterium tuberculosis (MTB). Current diagnostic criterias for TB infection includes 1) clinical symptoms, 2) radiography, and 3) positive culture results. Clinical tests should also be performed to monitor the effectiveness of treatment and to check whether patients have taken prescribed drugs on the regular basis or not.
In terms of clinical tests of TB at present, it only takes about 30~60 minutes to perform acid-fast stain, nevertheless, the sensitivity of this test is low. Bacterial culture is still considered as the gold standard of TB diagnosis, however, it takes up to 1-2 months to get culture results. Previous studies have indicated that individual infected by MTB may stimulate the expression of T helper cell, which in turn triggers Th1 and Th17 to produce varieties of cytokines and immune response of Treg cells to destroy MTB.
The purpose of this study is to investigate the differences of various cytokine levels in non-TB patients and TB patients, before and after anti-TB treatment. The difference in cytokine level patterns of these groups may be helpful in the diagnosis and evaluation of therapeutic response of TB patients.
Forty-four samples from 34 patients which includes 19 confirmed TB cases (pre-treatment status) and 10 confirmed TB cases (post-treatment status, after six-month anti-TB treatment), 5 latent TB cases and 10 healthy controls were recruited. The cytokine levels associated with Th1, Th2, Th17 and Treg of these 5 groups were measured, which includes IL-1β, IL4,IL-6, IL-12, IL-17A, IFN-γ, TNF-α and TGF-β1. Our results show that the cytokine levels of IL-6, IL-17A and IFN-γ are significantly higher in TB- confirmed cases comparing to healthy controls. 10 TB confirmed cases with six-month anti-TB treatment have higher IL12 and IL17A and lower TNF-α levels. The latent TB cases showed higher IL17A and TGF-β1 level but lower IL6 level comparing to the healthy controls.
IL17 levels are significantly higher in pre-treated TB patients, post-treated TB patients and latent TB patients comparing to healthy controls. IL17 concentration in serum therefore may be a disease indicator of tuberculosis infection. Furthermore, the decrease of IL17 concentration with time may be used for clinicians to evaluate the effectiveness of anti-TB treatment. It can also be used as a prognosis factor of TB patient. On the other hand, in cases of ineffective treatment of TB patients with persistent bacteria in sputum, a higher level of IL-6 was observed, it may thus be able to be a disease marker for more intensive therapeutic regimens and continuous patient isolation.
In conclusion, the increase of IL6 and IL17 level may be used to be disease markers for diagnosis, monitoring, treatment response and outcome of TB patient. They could also be indicators of open pulmonary tuberculosis and may be the potential disease markers for TB infection.

論文審定書 i
致謝 ii
中文摘要 iii
英文摘要 v
縮寫表 xi
第一章 緒論 1
1.1、認識TB菌 1
1.2、肺結核流行病學 2
1.3、結核分枝桿菌診斷之介紹 3
1.3.1、痰塗片之抗酸性細菌染色法(Acid fast stain)檢驗 4
1.3.2、Rapid test TB test (ICT) Identification Immuno- chromatographic test 4
1.3.3、Real-time PCR 5
1.4、肺結核感染與T helper cell免疫機轉 5
1.5、T helper cell分泌相關 cytokine機轉 6
第二章 研究動機 8
2.1、背景原因 8
2.2、本研究三大目標 8
第三章 材料與方法 9
3.1、潛伏性肺結核測試套組材料 9
3.1.1、Quantiferon-TB-Gold In-Tube test (QFT) 9
3.1.2、QFT TB Gold ELISA kit 9
3.2、潛伏性肺結核測試套組方法 9
3.2.1、第一階段–血液培養及血漿取得 9
3.2.2、第二階段–ELISA實驗 10
3.3、CYTOKINE分析CBA套組材料 10
3.4、CYTOKINE分析CBA套組方法 11
3.4.1、反應原理 11
3.4.2、反應步驟 11
3.5、實驗設計 12
3.5.1、檢體來源及收集個數 12
3.5.2、實驗進行 12
3.6、統計分析 13
第四章 結果 14
4.1、CYTOKINE結果分析 14
4.1.1、偵測相關CYTOKINE含量分析 14
4.1.1.1、偵測相關CYTOKINE含量分析-健康人組及治療前組兩組 14
4.1.1.2、偵測相關CYTOKINE含量分析-健康人組及治療後組兩組 15
4.1.1.3、偵測相關CYTOKINE含量分析-健康人組及潛伏性TB病人組兩組 15
4.1.1.4、偵測相關CYTOKINE含量分析-治療前組及治療後組兩組 16
4.1.2、檢定統計學數據分析 16
4.1.3、CRP與IL-6相關性比較 17
第五章 討論 18
第六章 結論與展望 22
參考文獻 24
 
表次

表一. Characteristic of The participants in four groups 28
表二-1. Comparison for acute phase protein and cytokine concentrations between healthy controls and TB patients 29
表二-2. Comparison for acute phase protein and cytokine concentrations between healthy controls and TB patients after treatment 29
表二-3. Comparison for acute phase protein and cytokine concentrations between healthy controls and latent TB patients 30
表二-4. Pair Comparisonbetween TB patients and TB patients after treatment of acute phase protein and cytokineconcentrations 30
表三-1. Comparison between serum levels of CRP and IL6 and bacterial counts in sputum of TB patients and healthy controls 31
表三-2. Comparison done using Spearman's rho Test 31
表四. 實驗結果與文獻查詢比較結果-16篇文獻 32
表五. CUT-OFF value of IL6、IL17A and IFN-γ for TB infection 33

圖次

圖1. TB治療前後、潛伏TB病人與健康人CYTOKINE表現直條圖分析比較 34
圖2. TB治療前後、潛伏TB病人與健康人CYTOKINE表現相關係數顯著性 35
圖3. 四組(0健康人，1治療前，6治療後，3潛伏TB)偵測相關CYTOKINE平均及差異 36
圖4. TB發病與否偵測，IL6、IL17A及IFNγ ROC curve 37

附件次

附件一-1. 人體試驗倫理委員會同意書IRB 99-3784B 38
附件一-2. 人體試驗倫理委員會同意書IRB 103-7025B 39
附件二-1. 檢驗方法補充-AFB 40
附件二-2. 檢驗方法補充-ICT 40
附件二-3. 檢驗方法補充-MALDI-TOF 41
附件三、 CYTOKINE結果分析-RAW DATA 42

附圖次
附圖1. 細菌感染之CYTOKINE分泌機轉 45
附圖2. Helper cells, well known subsets and their functions 46
附圖3. Cytokine network. 47
附圖4. Overview of the immune response in tuberculosis 48
附圖5. QFT TB Gold ELISA kit 49
附圖6. 流式細胞儀-實驗原理及上機 50

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