白色念珠菌胞外磷脂酶B在兔角膜真菌感染中毒力作用的研究  

Virulence of extraceHular phospholipase B of Candlda albicans in rabbit experimental keratomycosis

作　　者：马林[1] 谢立信[1] 董晓光[1] 史伟云[1] 

机构地区：[1]山东省眼科研究所山东省眼科学重点实验室-省部共建国家重点实验室培育基地,青岛266071

出　　处：《中华眼科杂志》2008年第3期237-243,共7页Chinese Journal of Ophthalmology

基　　金：国家自然科学基金资助项目（30630063,30271394）;山东省科技攻关计划项目（2004GG2202154）

摘　　要：目的探讨白色念珠菌胞外磷脂酶B（PLB）在兔真菌性角膜炎中的毒力作用。方法为实验研究。选用白色念珠菌PLB基因缺失株及其相应母体株，角膜接触镜法建立两种菌株兔角膜感染动物模型，通过对动物模型在2、3、5及8d不同时间点的临床观察和病变角膜的组织病理学检查，比较两种菌株的毒力；并建立两种菌株孢子与体外培养的兔角膜基质细胞共同孵育体系，通过扫描电镜、酶联免疫吸附实验及流式细胞仪检测，观察两种菌株对细胞的影响，确定PLB的毒力作用机制。结果两种菌株菌丝在角膜中均呈垂直方式生长；动物模型接种后2d，两种菌株菌丝在角膜中的浸润深度比较差异有统计学意义（P=0．002）；而在3、5及8d时问点的两种菌株菌丝浸润深度及所有时问点的兔角膜真菌感染的临床表现、角膜炎性反应程度比较差异均无统计学意义（P〉0．05）。与单层细胞共同孵育后，两种菌株孢子、菌丝的黏附率、菌丝生成率比较差异无统计学意义（P〉0．05），而破坏穿透细胞膜的菌丝数量比较差异有统计学意义（P=0．009）；角膜基质细胞加母体株孢子悬液组培养上清中前列腺素E2（PGE2）的含量为（65466±5773）pg／ml，较角膜基质细胞加缺失株孢子悬液组培养上清的PGE2含量（18025±5232）pg／ml明显升高；母体株组、缺失株组及正常对照组细胞膜被破坏细胞的百分比分别为3．02％、2．04％及0．12％；凋亡细胞百分比分别为33．17％、27．56％及1．46％；正常活细胞百分比分别为63．81％、70．40％及98．41％。结论白色念珠菌胞外PLB在兔角膜感染的整个病理损害中，仅在真菌黏附于角膜刚开始侵袭角膜细胞时有促进菌丝侵入细胞的作用，而在其后角膜炎性反应的病理损害中，并无明显的毒力作用。Objective To determine the virulence of extracellular phospholipase B （PLB） of Candida albicans in experimental keratomycosis. Methods It was an experimental study. The PLB-deficient mutant strain of Candida albicans and its isogenie parental strain were used in this study. The effects of these two strains on the model of experimental keratomyeosis in 48 New Zealand albino rabbits covered with contact lens was compared by observing the dynamic changes clinically and histopathologieally. In vitro, these two strains were incubated with the corneal stromal cells separately （37℃ , 5 % CO2 ）. The influence of these two strains on monolayer keratoeytes were detected by scanning electron microscopy （ SEM ） , enzyme linked immunosorbent assay （ELISA）, and flow cytometry with Annexin V/propidium iodide. Results The hyphae of these two strains grew perpendicularly to the corneal stromal lamellae. The difference of the hyphal invasion inoculated for 2 days by these two isogenic strains was statistically significant （ P = 0. 002） , but at other intervals, no significant difference was found. The severity of the inflammation in parental keratomycosis was the same as that in PLB null strain at any time points （ P 〉 0.05 ）. Under SEM, the morphogenesis and the number of adherent germ tubes of these two isogenie strains appeared similarly （ P 〉 0. 05 ） , but the number of germ tubes penetrating cell monolayer was significantly different （ P = 0. 009 ）. Obviously more prostaglandin E2 （ PGE2 ） was detected in the culture superuatants of parental strain group （65 466 ±5773） pg/ml than that of the null strain group （ 18 025 ±5232） pg/ml. The percentages of the cells with damaged cellular membrane in the parental group, the PLB null group and the control group, were 3.02%, 2. 04% and 0. 12%, respectively. The pereentages of apoptosis cells in these three groups were 33.17% , 27. 56% and 1.46%, respectively. The percentages of living cells were 63.81%, 70. 40% and 98.41%, respective

关 键 词：眼感染 真菌性 角膜炎 白色念珠菌 溶血磷脂酶 基因缺失 

分 类 号：R686[医药卫生—骨科学]