結核病長久以來一直是全世界致病及致死相當重要的傳染病之一。在臺灣地區結核病仍有非常高的盛行率。證據顯示宿主的基因會對結核分枝桿菌感染易感性造成影響。年幼兒童有如免疫缺陷患者，一旦感染會增加結核病發病及進展為肺外感染的危機。甘露聚糖結合凝集素 (mannose-binding lectin, MBL) 會以”不具抗體依賴性”(antibody-independent) 的方式活化補體系統，促進補體斡旋(complement-mediated) 吞噬作用，在先天性免疫反應(innate immunity) 調節單核球釋出發炎性細胞素過程中扮演重要的角色，亦是人類對感染症的易感性或對抗性的重要相關分子。根據文獻報導(大部份針對成年人) 結果顯示，低MBL產能相關的MBL結構基因exon 1上突變或起動子基因上的變異基因型，對結核菌感染有保護作用；但其他研究者則認為高MBL量才對結核菌感染具有保護作用。本研究主要目的在探討，患有頸部結核性淋巴節炎兒童對結核菌感染之易感性與MBL基因多型性的相關性。研究中收集 139位經組織病理學檢驗，並以耐酸性染色及PCR確認患有頸部結核性淋巴節炎的兒童，和102位對照健康者石蠟包埋檢體，以即時聚合酶鏈反應 (real-time PCR)檢驗法偵測其MBL基因exon 1及啟動子基因多型性，並以統計學方法分析其與結核病罹患感受性之相關性。結果顯示結核病患者(TB+)具有A基因型的比率明顯高於未患病的正常對照組(TB-)(82.7% vs 72.6%; odds ratio 1.813; p=0.007) 。MBL -550啟動子基因型在TB+ 及TB- 兩組間亦呈現有意義的差異，但卻相反的在低MBL產能的L/L基因型在TB+患者有較高的頻率(34.5% vs 21.6%; odds ratio 1.918; p=0.029)。 MBL -221啟動子基因型的頻率在TB+ 及TB- 兩組間未見有意義的差異。研究結果支持了MBL濃度可能會對結核菌易感性(susceptibility)造成影響的結論。

Tuberculosis (TB) is an important cause of morbidity and mortality worldwide. The high incidence is still found in Taiwan. There is strong evidence that host genes influence individual susceptibility to tuberculosis. Young children, like immunocompromised patients, once infected are at increased risk for TB disease and progression to extrapulmonary disease. Thus far, to identify the genes responsible for the variation in the human susceptibility/resistance to TB has remained elusive. Mannose-binding lectin (MBL) activates the complement system in an antibody-independent manner, enhances complement-mediated phagocytosis, and plays an important role in innate immunity in the regulation of inflammatory cytokine release by monocytes. It is one of the molecules that have been suggested to have a link to human susceptibility or protection against infection. According to some studies (mostly conducted in adult populations) , low levels of MBL associated with variant alleles at the promoter and exon 1 regions of MBL protect against tuberculosis. Other investigators instead claim that protection against the disease is associated with high levels of MBL. In this study we aimed to investigate the relationships between the susceptibility to TB and MBL gene polymorphisms in children with cervical mycobacterial lymphadenitis infected by M. tuberculosis.139 case patients with cervical mycobacterial lymphadenitis and 102 unrelated healthy control subjects were tested by real-time PCR for polymorphisms at the promoter and the exon 1 regions of the MBL gene. Diagnosis of mycobacterial lymphadenitis infected by M. tuberculosis, based on findings of pathological examination of the lymph nodes, was confirmed by acid-fast stain and TB PCR.The frequency of A allele was significantly higher in TB+ patients compared with TB- controls (82.7% vs 72.6%; odds ratio 1.813; p=0.007). The frequency of high-producer MBL2 genotypes (A/A) was higher in TB+ patients than in TB- subjects (70.5% vs 45.1%, odds ratio 2.91, p<0.001), while patients carried the B alleles (A/B and B/B) that have decreased levels of MBL was inversely associated with mycobacterial infectivity (29.5% vs 54.9%; odds ratio 2.910; p<0.001). The frequencies of MBL promoter -550 genotypes also revealed a significant difference between TB+ and TB- groups (p = 0.046), but in contrast, with significantly higher frequency of L/L genotype (of low MBL level) in TB+ patients (34.5% vs 21.6%; odds ratio 1.918; p=0.029). The frequencies of MBL promoter -221 genotypes (X and Y) was similar in TB+ and TB- groups.This study supports the conclusion that MBL can protect or predispose the host to tuberculosis, depending on the host’s haplotype pair.

目 錄
論文審定書…………………………………………………………… i
誌謝…………………………………………………………………… ii
中文摘要………………………………………………………….….. iii
英文摘要………………………………………..……………………. iv
第 一 章 簡介………………………………………..………………1
1.1結核病(Tuberculosis) …………………………1
1.1.1結核病流行概況…………………………………………1
1.1.2結核病的病原體和感染相關因素………………………2
1.1.3臨床表現……………………………………………………3
1.1.4實驗室檢驗方法……………………………………………4
1.2補體系統………………………………………………………5
1.2.1活化補體系統的三種路徑………………………………6
1.2.2 補體蛋白會協同吞噬細胞完成對病原體的破壞………7
1.3甘露聚糖結合凝集素(Mannose binding lectin，MBL) …8
1.3.1MBL與致病微生物相互作用………………………………9
1.3.2MBL2基因多型性…………………………………………9
1.3.3MBL2基因多型性與感染結核菌的相關性……………10
第 二 章 材料與方法………………………………………………11
2.1 研究設計與樣本………………………………………11
2.2 實驗原理及步驟………………………………………11
2.2.1 病理組織切片標本製作…………………………………11
2.2.2 石蠟塊組織DNA的萃取…………………………………12
2.2.3 TB PCR分析………………………………………………13
2.2.4 Real-Time PCR分析MBL genotyping…………………14
2.3統計分析…………………………………………………15
第 三 章 結果 ………………………………………………………16
第 四 章 討論 ………………………………………………………18
圖 ………………………………………………………………………22
表 ………………………………………………………………………25
參考文獻 ………………………………………………………………28
附錄…………………………………………………………………….34








圖　次
圖一.分枝桿菌在病理組織切片以Ziehl-Neelsen組織染色的型態
…………………………………………………………22
圖二.頸部淋巴結之組織切片以蘇木紫-伊紅染色顯微鏡下的型態…
………………………………………………………………23
圖三.TB PCR電泳結果………………………………………………24


























表　次
表一.MBL的基因型別和對偶基因在TB+病人及TB-個體中的表現…………………………………………………………………25
表二.MBL的半套體在TB+病人及TB-個體中的表現…………………26
表三.比較MBL基因套型HYA與結核菌感染之間的相關性………… 27














附　圖
附圖一.補體系統活化的三種路徑………………………………34
附圖二.MBL補體活化過程 ……………………………………………35
附圖三.MBL2基因的結構及甘露聚糖結合凝集素立體結構…………36
附圖四.甘露聚糖結合凝集素(MBL)的基因型及基因多型性 ………37
附圖五.Real-time PCR反應原理……………………………………38

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