烟曲霉菌对细胞的侵染机制及念珠菌PCR-SSCP分子流行病学技术的初步研究

发布时间：2018-03-25 00:30

本文选题：烟曲霉　切入点：光滑念珠菌　出处：《西北大学》2008年硕士论文

【摘要】： 目的: 研究探讨烟曲霉孢子侵染宿主肺上皮细胞以及被巨噬细胞吞噬过程中宿主细胞内PLD活性变化及其对烟曲霉侵染率的影响规律,并初步分析该内化吞噬过程中烟曲霉孢子、肌动蛋白蛋白骨架以及PLD蛋白在时间及空间上的相互关系。另外,论文还将探讨PCR-SSCP技术用于真菌分子流行病学研究的可行性及最适条件。方法: 以烟曲霉孢子刺激人肺Ⅱ型上皮细胞(A549)和鼠巨噬细胞(J774.A1),测定不同感染比(MOI)和不同感染时间下,两种细胞内PLD活性以及烟曲霉孢子侵染效率的变化规律;用PLD化学抑制剂预处理细胞,测定其对烟曲霉孢子侵染效率的影响;利用免疫荧光标记以及激光共聚焦显微镜等技术,观察烟曲霉孢子在侵染过程中与宿主细胞肌动蛋白骨架和PLD蛋白的空间定位关系;对来自某两家医院的19株光滑念珠菌进行PCR-SSCP多态性分型分析,对扩增目标区域、电泳的条件,如温度、甘油浓度等进行比较分析。结果: 烟曲霉孢子对A549细胞的侵染率随感染比和感染时间的增加而显著增加,并伴有PLD的显著激活;而对J774.A1细胞的侵染率随感染时间的延长而降低;两种侵染过程中均发生细胞肌动蛋白骨架的剧烈重排,在烟曲霉孢子周围形成吞噬杯,而PLD蛋白也可能在被刺激后移位至吞噬杯周围;PLD抑制剂1-丁醇预处理两种细胞均可显著降低烟曲霉孢子对细胞的侵染率;PCR-SSCP用于临床念珠菌分子流行病学分析较为合适的条件为:对ITSⅠ区进行扩增并在15℃及5%甘油存在的条件下进行SSCP电泳。结论: 在烟曲霉孢子侵染宿主细胞过程中伴有PLD的激活和细胞肌动蛋白骨架的重排,二者在空间上的定位密切相关;PLD活性的降低对烟曲霉侵染率有抑制作用;在合适的条件下,PCR-SSCP技术是一种快速、简便的适合于念珠菌分型的分子流行病学研究手段。
[Abstract]:Objective:
Study on Aspergillus fumigatus infection of lung epithelial cells and macrophage PLD activity changes in host cells in the process and the influence law of Aspergillus fumigatus infection rate, and preliminary analysis of the internalization in the phagocytic process of Aspergillus fumigatus spores, the relationship between actin protein backbone and PLD protein in time and space. In addition, the paper also will to investigate the feasibility of PCR-SSCP technology for fungal molecular epidemiology studies and the optimum conditions.
Method:
The spores of Aspergillus fumigatus stimulate human lung type II epithelial cells (A549) and rat macrophages (J774.A1), the determination of different infection than (MOI) and different time of infection, two kinds of intracellular PLD activity and the variation of spores of Aspergillus fumigatus infection efficiency; using PLD chemical inhibitors pretreatment of cells to study its effects on Aspergillus fumigatus the efficiency of infection; using immunofluorescence and confocal laser microscopy, observe the relationship between spatial location of Aspergillus fumigatus spores in the infection process and host cell actin cytoskeleton and PLD protein; from two hospitals of 19 strains of Candida glabrata were analyzed PCR-SSCP polymorphism, the amplification of the target area, electrophoresis the conditions, such as temperature, concentration of glycerol were compared and analyzed.
Result:
Aspergillus fumigatus infection rate of A549 cells with the infection ratio and increase the time of infection and increased significantly, and was accompanied by activation of PLD; and the infection rate of J774.A1 cells decreased with time of infection; dramatic rearrangement of actin cytoskeleton were two kinds of infection process, the phagocytic cup formed around Aspergillus fumigatus the spores, PLD protein may also shift to the phagocytic cup around when it was stimulated; PLD inhibitor 1- n-butanol pretreatment two cells could significantly decrease the spores of Aspergillus fumigatus infection on cell rate; PCR-SSCP for molecular epidemiological analysis of clinical Candida suitable condition: ITS 1 and SSCP were amplified in electrophoresis there are 15 DEG C and 5% glycerol conditions.
Conclusion:
The spores of Aspergillus fumigatus infecting a host cell process with PLD activation and actin cytoskeleton rearrangement, two closely related position in space; the decrease of the activity of PLD of Aspergillus fumigatus infection rate of inhibition; under suitable conditions, the PCR-SSCP technique is a rapid, easy means for molecular epidemiological study albicans type.

【学位授予单位】：西北大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R379

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