本文關(guān)鍵詞： 煙曲霉 膠霉毒素 支氣管上皮細(xì)胞　出處：《福建醫(yī)科大學(xué)》2013年碩士論文　論文類型：學(xué)位論文

【摘要】：目的：通過建立煙曲霉感染支氣管上皮細(xì)胞的體外模型，以探討膠霉毒素在煙曲霉感染中的作用及其機(jī)制。 方法：1、觀察膠霉毒素（gliotoxin GT）對(duì)支氣管上皮細(xì)胞（human bronchialepithelial cells HBE）凋亡和細(xì)胞間粘附分子（ICAM-1）表達(dá)的影響：1）藥物配制：膠霉毒素樣品先溶于95%乙醇中，再用培養(yǎng)基（DMEM）稀釋到所需濃度；2）細(xì)胞接種：將野生型支氣管上皮細(xì)胞以密度為4000/ml接種于直徑9cm培養(yǎng)皿中，每皿5ml，并設(shè)置對(duì)照組；3）加藥：分別設(shè)置3個(gè)濃度，當(dāng)細(xì)胞貼壁48h時(shí)（孔底細(xì)胞長(zhǎng)滿90%以上），加入新鮮含藥培養(yǎng)基5ml，后放入CO_2培養(yǎng)箱中培養(yǎng)24h。4)結(jié)果的測(cè)定：藥物作用24h后，收集不同濃度組細(xì)胞及提取各組細(xì)胞的總RNA，采用流式細(xì)胞儀和RT-PCR檢測(cè)細(xì)胞凋亡和表達(dá)ICAM-1和NF-κB的變化。2、建立煙曲霉感染支氣管上皮細(xì)胞的體外模型。1)菌株的培養(yǎng)：將煙曲霉菌接種到PDA瓊脂平板，置于30℃培養(yǎng)箱內(nèi)培養(yǎng)3-4天，連續(xù)活化兩次，PBS重懸孢子并計(jì)數(shù)。2)野生型人支氣管上皮細(xì)胞（HBE）的培養(yǎng)：將支氣管上皮細(xì)胞以密度為4000/ml接種于直徑9cm培養(yǎng)皿中，，每皿加入5ml細(xì)胞懸液，后放入CO_2培養(yǎng)箱中培養(yǎng)48h。3)共培養(yǎng)模型的建立：當(dāng)細(xì)胞密度為90%以上時(shí)，按1:1000（菌孢子數(shù):支氣管上皮細(xì)胞數(shù)）加入菌懸液，37℃、5%CO_2條件下培養(yǎng)2h，PBS沖洗，棄上清液，加入一定量的DMEM完全培養(yǎng)基，置于細(xì)胞培養(yǎng)箱中，繼續(xù)培養(yǎng)。4)結(jié)果的測(cè)定：分別于共培養(yǎng)12、24、36h終止實(shí)驗(yàn)以獲取菌絲、支氣管上皮細(xì)胞和12、24、36h提取細(xì)胞培養(yǎng)上清液，采用顯微鏡觀察、流式細(xì)胞儀（Annexin V-FITC法）和RT-PCR等方法，檢測(cè)真菌表達(dá)膠霉毒素的水平、支氣管上皮細(xì)胞凋亡和ICAM-1的表達(dá)情況。 結(jié)果： 1. RT-PCR檢測(cè)HBE凋亡基因表達(dá)的結(jié)果顯示：膠霉毒素處理后，細(xì)胞凋亡基因Bak和Bax均表達(dá)上升（p0.005和p0.05），特別Bak基因表達(dá)最明顯，且與藥物濃度呈一定相關(guān)性； 2、真菌RT-PCR的結(jié)果顯示：與對(duì)照組（煙曲霉孢子狀態(tài)）相比，模型組中12h和24h時(shí)間點(diǎn)真菌膠霉毒素的表達(dá)沒有統(tǒng)計(jì)學(xué)意義（P0.01），而在36h時(shí)間點(diǎn)時(shí)真菌膠霉毒素表達(dá)明顯升高（P0.01），有統(tǒng)計(jì)學(xué)意義。 3、支氣管上皮細(xì)胞凋亡基因的RT-PCR結(jié)果顯示：與對(duì)照組（支氣管上皮細(xì)胞單獨(dú)培養(yǎng)時(shí)）相比，細(xì)胞凋亡基因從24h時(shí)間點(diǎn)開始表達(dá)上調(diào)，到36h時(shí)間點(diǎn)到一個(gè)高峰，特別是凋亡基因Bak，表達(dá)異常明顯（p0.05）。同時(shí)，抑凋亡基因Bcl-2表達(dá)下調(diào)，在36h時(shí)間點(diǎn)下調(diào)有明顯統(tǒng)計(jì)學(xué)意義（p0.05）。 結(jié)論： 1、膠霉毒素在煙曲霉感染中發(fā)揮著重要的細(xì)胞毒性作用。 2、膠霉毒素可能主要通過誘導(dǎo)支氣管上皮細(xì)胞凋亡來(lái)發(fā)揮細(xì)胞毒性作用。 3、膠霉毒素對(duì)支氣管上皮細(xì)胞免疫反應(yīng)的抑制可能有濃度依賴性。
[Abstract]:Aim: to establish a model of bronchoepithelial cells infected by Aspergillus fumigatus in vitro to investigate the role of collotoxin in the infection of Aspergillus fumigatus and its mechanism. Method 1. To observe the effect of gliotoxin GTZ on human bronchialepithelial cells HBE in bronchial epithelial cells. Effects of apoptosis and ICAM-1 expression on the expression of ICAM-1) the drug preparation: the colloidal toxin sample was first dissolved in 95% ethanol. Then DMEM was diluted to the desired concentration; 2) Cell inoculation: the wild-type bronchial epithelial cells were inoculated in 9cm culture dish with density of 4000ml / ml, 5 ml per dish, and the control group was set up. 3) Additives: three concentrations were set up, when the cells adhered to the cell wall for 48 hours (the cells at the bottom of the pore grew up to 90% or more), and the fresh drug containing medium was added to the culture medium of 5ml. The results of 24 h culture in CO_2 incubator: after 24 hours of treatment, the cells of different concentration groups were collected and the total RNA of each group was extracted. Apoptosis and expression of ICAM-1 and NF- 魏 B were detected by flow cytometry and RT-PCR. 2. In vitro culture of Aspergillus fumigatus infected bronchial epithelial cells: Aspergillus fumigatus was inoculated into PDA Agar plate and cultured in 30 鈩

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