探讨胶霉毒素在支气管烟曲霉感染中的作用

发布时间：2018-01-31 23:40

  本文关键词： 烟曲霉 胶霉毒素 支气管上皮细胞　出处：《福建医科大学》2013年硕士论文　论文类型：学位论文

【摘要】：目的：通过建立烟曲霉感染支气管上皮细胞的体外模型，以探讨胶霉毒素在烟曲霉感染中的作用及其机制。 方法：1、观察胶霉毒素（gliotoxin GT）对支气管上皮细胞（human bronchialepithelial cells HBE）凋亡和细胞间粘附分子（ICAM-1）表达的影响：1）药物配制：胶霉毒素样品先溶于95%乙醇中，再用培养基（DMEM）稀释到所需浓度；2）细胞接种：将野生型支气管上皮细胞以密度为4000/ml接种于直径9cm培养皿中，每皿5ml，并设置对照组；3）加药：分别设置3个浓度，当细胞贴壁48h时（孔底细胞长满90%以上），加入新鲜含药培养基5ml，后放入CO_2培养箱中培养24h。4)结果的测定：药物作用24h后，收集不同浓度组细胞及提取各组细胞的总RNA，采用流式细胞仪和RT-PCR检测细胞凋亡和表达ICAM-1和NF-κB的变化。2、建立烟曲霉感染支气管上皮细胞的体外模型。1)菌株的培养：将烟曲霉菌接种到PDA琼脂平板，置于30℃培养箱内培养3-4天，连续活化两次，PBS重悬孢子并计数。2)野生型人支气管上皮细胞（HBE）的培养：将支气管上皮细胞以密度为4000/ml接种于直径9cm培养皿中，，每皿加入5ml细胞悬液，后放入CO_2培养箱中培养48h。3)共培养模型的建立：当细胞密度为90%以上时，按1:1000（菌孢子数:支气管上皮细胞数）加入菌悬液，37℃、5%CO_2条件下培养2h，PBS冲洗，弃上清液，加入一定量的DMEM完全培养基，置于细胞培养箱中，继续培养。4)结果的测定：分别于共培养12、24、36h终止实验以获取菌丝、支气管上皮细胞和12、24、36h提取细胞培养上清液，采用显微镜观察、流式细胞仪（Annexin V-FITC法）和RT-PCR等方法，检测真菌表达胶霉毒素的水平、支气管上皮细胞凋亡和ICAM-1的表达情况。 结果： 1. RT-PCR检测HBE凋亡基因表达的结果显示：胶霉毒素处理后，细胞凋亡基因Bak和Bax均表达上升（p0.005和p0.05），特别Bak基因表达最明显，且与药物浓度呈一定相关性； 2、真菌RT-PCR的结果显示：与对照组（烟曲霉孢子状态）相比，模型组中12h和24h时间点真菌胶霉毒素的表达没有统计学意义（P0.01），而在36h时间点时真菌胶霉毒素表达明显升高（P0.01），有统计学意义。 3、支气管上皮细胞凋亡基因的RT-PCR结果显示：与对照组（支气管上皮细胞单独培养时）相比，细胞凋亡基因从24h时间点开始表达上调，到36h时间点到一个高峰，特别是凋亡基因Bak，表达异常明显（p0.05）。同时，抑凋亡基因Bcl-2表达下调，在36h时间点下调有明显统计学意义（p0.05）。 结论： 1、胶霉毒素在烟曲霉感染中发挥着重要的细胞毒性作用。 2、胶霉毒素可能主要通过诱导支气管上皮细胞凋亡来发挥细胞毒性作用。 3、胶霉毒素对支气管上皮细胞免疫反应的抑制可能有浓度依赖性。
[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

本文编号：1480306