本文選題：煙曲霉　切入點(diǎn)：光滑念珠菌　出處：《西北大學(xué)》2008年碩士論文

【摘要】： 目的: 研究探討煙曲霉孢子侵染宿主肺上皮細(xì)胞以及被巨噬細(xì)胞吞噬過(guò)程中宿主細(xì)胞內(nèi)PLD活性變化及其對(duì)煙曲霉侵染率的影響規(guī)律,并初步分析該內(nèi)化吞噬過(guò)程中煙曲霉孢子、肌動(dòng)蛋白蛋白骨架以及PLD蛋白在時(shí)間及空間上的相互關(guān)系。另外,論文還將探討PCR-SSCP技術(shù)用于真菌分子流行病學(xué)研究的可行性及最適條件。 方法: 以煙曲霉孢子刺激人肺Ⅱ型上皮細(xì)胞(A549)和鼠巨噬細(xì)胞(J774.A1),測(cè)定不同感染比(MOI)和不同感染時(shí)間下,兩種細(xì)胞內(nèi)PLD活性以及煙曲霉孢子侵染效率的變化規(guī)律;用PLD化學(xué)抑制劑預(yù)處理細(xì)胞,測(cè)定其對(duì)煙曲霉孢子侵染效率的影響;利用免疫熒光標(biāo)記以及激光共聚焦顯微鏡等技術(shù),觀察煙曲霉孢子在侵染過(guò)程中與宿主細(xì)胞肌動(dòng)蛋白骨架和PLD蛋白的空間定位關(guān)系;對(duì)來(lái)自某兩家醫(yī)院的19株光滑念珠菌進(jìn)行PCR-SSCP多態(tài)性分型分析,對(duì)擴(kuò)增目標(biāo)區(qū)域、電泳的條件,如溫度、甘油濃度等進(jìn)行比較分析。 結(jié)果: 煙曲霉孢子對(duì)A549細(xì)胞的侵染率隨感染比和感染時(shí)間的增加而顯著增加,并伴有PLD的顯著激活;而對(duì)J774.A1細(xì)胞的侵染率隨感染時(shí)間的延長(zhǎng)而降低;兩種侵染過(guò)程中均發(fā)生細(xì)胞肌動(dòng)蛋白骨架的劇烈重排,在煙曲霉孢子周圍形成吞噬杯,而PLD蛋白也可能在被刺激后移位至吞噬杯周圍;PLD抑制劑1-丁醇預(yù)處理兩種細(xì)胞均可顯著降低煙曲霉孢子對(duì)細(xì)胞的侵染率;PCR-SSCP用于臨床念珠菌分子流行病學(xué)分析較為合適的條件為:對(duì)ITSⅠ區(qū)進(jìn)行擴(kuò)增并在15℃及5%甘油存在的條件下進(jìn)行SSCP電泳。 結(jié)論: 在煙曲霉孢子侵染宿主細(xì)胞過(guò)程中伴有PLD的激活和細(xì)胞肌動(dòng)蛋白骨架的重排,二者在空間上的定位密切相關(guān);PLD活性的降低對(duì)煙曲霉侵染率有抑制作用;在合適的條件下,PCR-SSCP技術(shù)是一種快速、簡(jiǎn)便的適合于念珠菌分型的分子流行病學(xué)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.

【學(xué)位授予單位】：西北大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R379

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本文編號(hào)：1660760