本文選題：結核分枝桿菌 + 自噬��； 參考：《第四軍醫(yī)大學》2012年碩士論文

【摘要】：結核分枝桿菌（Mycobacterium tuberculosis，MTb）自1882年被德國細菌學家證實可以引起肺結核以來，已致使世界三分之一的人感染，每年有170萬人死于結核病[1]。死亡人數(shù)僅亞于艾滋病，位居單一傳染病的第二位。極大影響了公眾健康。研究證實，肝素結合血凝素（heparin-bindinghemagglutinin adhesin，HBHA）是結核分枝桿菌最主要的粘附分子之一，分子量為28kDa，主要表達于細菌的表面，可結合肝素、硫化右旋糖酐等[2]。它有三個結構功能域，但其在細菌黏附和侵襲肺泡上皮細胞過程中的具體作用未明。 肺泡Ⅱ型上皮細胞是一組具有肺泡保護活性的細胞，同時也可合成和分泌表面活性劑和細胞因子[3]，在天然免疫過程中發(fā)揮重要的調節(jié)作用。而本課題組在之前的研究中證實，在結核分枝桿菌感染肺泡Ⅱ型上皮細胞A549細胞系后，能夠抑制A549細胞的自噬作用，結核分枝桿菌中的粘附素HBHA蛋白能夠抑制LC3基因的表達，很可能借此阻礙A549細胞利用自噬作用清除結核分枝桿菌。 為進一步闡明HBHA蛋白抑制LC3表達作用的機制，以及由此產生的促進結核菌感染的效應，本課題組分別合成了重組HBHA全長片段、C端缺失片段和N端缺失片段，并分別作用于肺泡Ⅱ型上皮細胞A549細胞系，觀察其自噬相關基因的表達變化、以及細胞因子釋放譜，從而初步闡明HBHA蛋白在MTb感染肺泡Ⅱ型上皮細胞后對其免疫調節(jié)作用的影響，探討其作用機制。具體研究如下： 1、HBHA蛋白純化、提取并對A549細胞自噬相關基因以及BCG感染效應的影響 本研究對已保存于細菌中的重組HBHA蛋白的全長片段、C端缺失片段和N端缺失片段進行表達、純化、提取。進行SDS凝膠電泳確定蛋白準確性和純度。 利用這三段蛋白片段分別作用于A549細胞，利用Western-blot驗證其干預LC3和Atg5的表達，以初步闡明HBHA蛋白抑制A549細胞自噬的信號通路。結果發(fā)現(xiàn)，三段蛋白均能抑制LC3的表達，但并不能干預Atg5的表達，提示HBHA蛋白不能影響Atg5㧟Atg12復合物的形成，卻可以抑制LC3復合物的形成，從而抑制A549細胞的自噬保護作用。 為驗證自噬與A549細胞抵抗結核分枝桿菌感染之間的作用，本研究利用BCG作用A549細胞與重組HBHA全長片段、C端缺失片段和N端缺失片段分別與BCG聯(lián)合后感染A549細胞進行比較，檢測其LDH釋放量。結果顯示，，加入蛋白后其LDH的釋放量明顯增加。證明抑制自噬后，細胞清除MTb的能力下降，保護作用減弱。為進一步確認HBHA蛋白在MTb感染A549細胞中的作用，本實驗利用BCG和HBHA抗體共同作用A549細胞，檢測LDH的釋放量。結果顯示，加入抗體組比只加入BCG感染A549細胞組的LDH釋放量顯著降低。說明利用抗體封閉HBHA蛋白后，MTb對A549細胞的破壞作用減弱，推測其HBHA抗體在MTb侵襲A549細胞的過程中具有一定的保護作用。 2、檢測HBHA蛋白作用細胞后細胞因子的分泌情況 HBHA蛋白抑制自噬的同時，是否參與了炎癥反應呢？本研究利用重組HBHA蛋白作用A549細胞，利用ELISA檢測其促炎細胞因子IL-1β、IL-6、IL-10、TNF-α、IFN-γ，結果發(fā)現(xiàn)A549細胞能分泌IL-1β、IL-6、IL-10、TNF-α、IFN-γ五種細胞因子，重組HBHA全長片段能促進A549細胞分泌IL-1β、IL-6、TNF-α、IFN-γ，而對于IL-10分泌沒有影響。 因此，本研究顯示，HBHA蛋白通過其核心α-螺旋結構的作用，抑制了LC3的表達及成熟，從而抑制了自噬,而抗HBHA抗體可以抵消這一作用。同時HBHA可引起炎癥因子IL-1β、INF-γ的釋放。從而提示，HBHA在MTb感染過程中具有重要的作用，是MTb菌感染、播散以及引起炎性反應的重要毒力因子。
[Abstract]:Mycobacterium tuberculosis ( MTb ) has been infected by a third of the world ' s people since the German bacteriologist confirmed that tuberculosis had been able to cause tuberculosis , and 1.7 million people died of tuberculosis every year . The death toll is only in the second place of the single infectious disease , which affects the public health . The study confirms that heparin - bindin - maglobadhesin ( HBHA ) is one of the most important adhesion molecules of Mycobacterium tuberculosis , with a molecular weight of 28 kDa , which is mainly expressed on the surface of bacteria , which can be combined with heparin , dextran and the like . It has three structural functional domains , but its specific role in bacterial adhesion and invasion of alveolar epithelial cells is unclear .

The alveolar type II epithelial cells are a group of cells with the activity of alveolus protection , but also can synthesize and secrete surface active agents and cytokines , and play an important role in regulating the natural immune process .

In order to further elucidate the mechanism of inhibiting LC3 expression by HBHA protein , and the effect of promoting the infection of mycobacterium tuberculosis , we synthesized the recombinant HBHA full - length fragment , the C - terminal deletion fragment and the N - terminal deletion fragment , respectively , and observed the changes of the expression of the autophagy - related gene and the cytokine release profile respectively . The effect of the HBHA protein on the immune regulation after MTb infection of alveolar type II epithelial cells was observed , and the mechanism of action was discussed .

1 . Purification and extraction of HBHA protein and its effect on the autophagy - related gene and BCG infection in A549 cells

In this study , the full - length fragment , C - terminal deletion fragment and N - terminal deletion fragment of recombinant HBHA protein which have been stored in bacteria were expressed , purified and extracted . The accuracy and purity of protein were determined by SDS gel electrophoresis .

The expression of LC3 and Atg5 was confirmed by Western - blot . The results showed that the three - segment protein could inhibit the expression of LC3 , but did not interfere with the expression of Atg5 , suggesting that HBHA protein could not interfere with the formation of Atg5 ? Atg12 complex , but could inhibit the formation of Atg5 ? Atg12 complex , thus inhibiting the autophagy protection of A549 cells .

In order to verify the effect of autophagy and A549 cells against Mycobacterium tuberculosis infection , the release amount of LDH in A549 cells was detected by BCG and HBHA , respectively .

2 . Detection of cytokine secretion after the action of HBHA protein

The results showed that A549 cells could secrete IL - 1尾 銆

本文編號：1951144