本文選題：結(jié)核病 + 單核苷酸多態(tài)性��； 參考：《吉林農(nóng)業(yè)大學(xué)》2013年碩士論文

【摘要】：背景：結(jié)核病是由結(jié)核分枝桿菌引起的一種慢性消耗性傳染病。引起該病的結(jié)核分枝桿菌作為一種細(xì)胞內(nèi)病原體,主要通過感染肺臟中具有吞噬功能的抗原遞呈細(xì)胞進(jìn)而侵入機體,雖然全球結(jié)核桿菌的攜帶者眾多,但是感染發(fā)病并發(fā)展成活動性結(jié)核病的僅僅占攜帶者的5%-10%。個體的遺傳因素和差異是影響和增加結(jié)核病發(fā)生以及發(fā)展的重要因素,同時也與結(jié)核病的易感性顯著相關(guān)。CD14,即脂多糖的受體,能通過脂多糖結(jié)合蛋白與脂多糖產(chǎn)生特異性結(jié)合,同時通過Toll樣受體4以及髓樣分化蛋白2向下游遞呈活化信號,通過釋放TNF-α、IL-1和IL-6等多種炎癥細(xì)胞因子,引發(fā)炎癥反應(yīng)的發(fā)生,在介導(dǎo)炎癥反應(yīng)過程和初次免疫應(yīng)答中起到重要的作用。目前研究發(fā)現(xiàn)CD14基因多態(tài)性位點與結(jié)核病易感性有關(guān),通過分析CD14基因多態(tài)性及其與患者血清中特異性抗體含量高低的相關(guān)性對于深入了解結(jié)核病發(fā)病機制、尋找新的檢測手段有著重要的意義。 目的：CD14基因啟動子區(qū)的多態(tài)性與結(jié)核病的發(fā)生有一定的聯(lián)系,但CD14基因多態(tài)性與患者血清中特異性抗體水平之間是否存在相關(guān)性,迄今尚無報道。本研究選擇CD14基因中的兩個多態(tài)性位點,采用直接測序的方法篩選出突變位點并對其多態(tài)性進(jìn)行分析,確定兩者之間是否存在相關(guān)性及與結(jié)核病易感性的聯(lián)系,為結(jié)核病的早期預(yù)防、診斷以及治療方面提供了一定的依據(jù),也為如何區(qū)分一些結(jié)核高風(fēng)險人群提供新的思路。 方法：運用ELISA的方法篩選出陽性和陰性血液樣本。按其數(shù)值高低各選取10個血液樣本提取基因組DNA；通過直接測序的方法篩選出C-159T和G-1145A的突變位點,分析其突變位點及其頻率與結(jié)核病易感性是否相關(guān)；通過分離血液樣本中的單個核細(xì)胞,并對其進(jìn)行免疫熒光染色,觀察細(xì)胞表面CD14抗原的表達(dá)量是否與結(jié)核病特異性抗體檢測結(jié)果之間的聯(lián)系。 結(jié)果：成功檢測出陽性血清樣本132個,陰性血清樣本250個；測序結(jié)果在C-159T基因中共發(fā)現(xiàn)10個突變位點,陽性血液樣本中出現(xiàn)了3個突變位點,突變頻率分別為12.5%、12.5%、75%,陰性血液樣本中出現(xiàn)了8個突變位點,突變頻率除一個突變位點為80%外,其他突變位點均為10%；在G-1145A基因中共發(fā)現(xiàn)8個突變位點,其中陽性血液樣本中出現(xiàn)了6個突變位點,突變頻率其中4個位點為11.1%,另外兩個位點為66.7%和88.9%,陰性血液樣本中出現(xiàn)了4個突變位點,突變頻率其中兩個位點為11.1%,另外兩個位點為66.7%和77.8%；成功分離得到單個核細(xì)胞,免疫熒光染色結(jié)果陰性血液樣本的細(xì)胞表面CD14抗原的表達(dá)量高于陽性血液樣本。 結(jié)論：發(fā)現(xiàn)CD14基因中的兩個多態(tài)性位點C-159T和G-1145A在陰性和陽性血液樣本中存在相同的突變位點,其突變頻率分別為77.8%和83.3%,而這兩個突變位點和報道過與結(jié)核病易感基因相關(guān)的突變位點相符,表明CD14基因的多態(tài)性與血清中PPD抗體的高低之間無明顯的相關(guān)性,說明CD14基因C-159T和G-1145A兩個多態(tài)性位點只能代表機體易感染結(jié)核病,但并不代表機體一定會發(fā)病。在G-1145A基因的突變位點中,第44個堿基位置在陽性血液樣本和陰性血液樣本中均出現(xiàn)了堿基缺失的突變現(xiàn)象,這個位點的堿基缺失目前還沒有報道,可能是一個新的潛在的多態(tài)性位點；在細(xì)胞表面CD14抗原的表達(dá)量上與PPD抗體量高低存在著負(fù)相關(guān)性,這說明外周血單個核細(xì)胞表面CD14抗原表達(dá)量與PPD抗體量高低存在著負(fù)相關(guān)性。結(jié)核病患者CD14的表達(dá)量會降低。
[Abstract]:Background : Tuberculosis is a kind of chronic consumptive infectious disease caused by Mycobacterium tuberculosis . Mycobacterium tuberculosis is a kind of intracellular pathogen .


Objective : The polymorphism of CD14 gene promoter region is associated with the occurrence of tuberculosis , but there is no correlation between CD14 gene polymorphism and the level of specific antibody in serum .


Methods : Positive and negative blood samples were screened by ELISA . Genomic DNA was extracted from 10 blood samples .
The mutation sites of C - 159T and G - 1145A were screened by direct sequencing .
By separating individual nuclear cells in the blood sample and performing immunofluorescence staining on it , it was observed whether the expression amount of the CD14 antigen on the surface of the cell was related to the detection result of the tuberculosis specific antibody .


Results : 132 positive serum samples and 250 negative serum samples were successfully detected .
The results showed that there were 10 mutation sites in C - 159T gene , 3 mutation sites appeared in positive blood samples , 12.5 % , 12.5 % , 75 % respectively , 8 mutation sites appeared in negative blood samples , and the mutation frequency was 80 % except for one mutation site and 10 % for other mutation sites .
Eight mutation sites were found in the G - 1145A gene , 6 mutation sites appeared in the positive blood samples , the mutation frequency was 11.1 % , the other two sites were 66.7 % and 89.9 % , 4 mutation sites appeared in the negative blood samples , the mutation frequencies were 11.1 % , and the other two sites were 66.7 % and 77.8 % ;
The expression of CD14 antigen in the cell surface of the negative blood sample was higher than that of the positive blood sample .


Conclusion : Two polymorphic sites C - 159T and G - 1145A in CD14 gene were found to have the same mutation sites in negative and positive blood samples . The mutation frequencies were 77.8 % and 83.3 % , respectively .
There was a negative correlation between the expression level of CD14 antigen and the amount of PPD antibody on the surface CD14 antigen in peripheral blood , suggesting a negative correlation between the expression of CD14 antigen and the amount of PPD antibody in peripheral blood mononuclear cells .
【學(xué)位授予單位】：吉林農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2013
【分類號】：R378.911

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