隐球菌感染体外血脑屏障模型的构建与应用

摘要

Objective To establish an in vitro Blood⁃Brain⁃Barrier (BBB) model,which was then applied to evaluate the capaci⁃ties of different cryptococcal strains in crossing Blood⁃Brain⁃Barrier.Methods Mouse brain endothelium cell bEND.3 was used to construct the Blood⁃Brain⁃Barrier model in vitro.In order to test its feasibility,we next examined the efficacy of several cryptococcal strains with different serotype or gene mutation in central nervous system (CNS) invasion.Results The transendothelial electrical resistance (TEER) measurement suggested that BBB model was constructed successfully.Compared with C.neoformans serotype A strain H99,JEC21 (C.neoformans serotype D) displayed a reduced efficacy in BBB invasion while the negative control strain (non pathogenic Saccharomyces cerevisiae strain Y187) with the lowest efficacy.Furthermore,the ure1 mutant (urease deletion) and the cap59 mutant (capsule deletion) showed a 50% and 80% decrease of their efficacy in crossing BBB compared to their parent strain H99,respectively (P<0.001).Conclusion The in vitro model of cryptococcal CNS infection was established successfully.Se⁃rotype,urease,and capsule was essential for C.neoformans to cross the Blood⁃Brain⁃Barrier.%目的：构建体外血脑屏障模型，并检测隐球菌不同菌株穿越血脑屏障的能力。方法本研究应用商品化的小鼠脑微血管内皮细胞系 bEND．3构建体外血脑屏障模型，并验证该模型应用于隐球菌穿越血脑屏障机制研究的可行性。通过构建模型，以非致病性的酿酒酵母作为阴性对照，比较新生隐球菌不同血清型标准株及基因缺陷株穿越体外血脑屏障能力的差异。结果跨膜电阻值（TEER）检测提示体外血脑屏障模型构建成功。检测结果显示酿酒酵母作为阴性对照穿越血脑屏障效率最低，新生隐球菌血清 A 型标准株 H99穿越细胞屏障效率最强，血清 D 型标准株 JEC21穿越细胞屏障效率显著低于 H99。较之 H99，黑色素酶缺陷株 lac1穿越体外血脑屏障模型的效率没有显著差异；尿素酶缺陷株 ure1效率显著下降（P＜0．05），约为标准株 H99通过率的59．9％；荚膜缺陷株 cap59突破体外血脑屏障模型效率最低，约为标准株 H99的18％（P＜0．001）。结论隐球菌中枢系统感染体外模型成功构建。新生隐球菌突破血脑屏障的能力与其血清型以及荚膜、尿素酶等毒力因子的表达密切相关。