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中国真菌学杂志 2015, Vol. 10  Issue (2): 92-95.

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隐球菌感染体外血脑屏障模型的构建与应用

孟云芳, 法振宗, 方伟, 周兆婧, 伊九, 顾菊林, 廖万清   

  1. 上海市医学真菌分子生物学重点实验室 上海市医学真菌研究所 上海长征医院皮肤科, 上海 200003
  • 收稿日期:2015-01-22 出版日期:2015-04-28 发布日期:2015-04-28
  • 通讯作者: 廖万清,E-mail:liaowanqing@sohu.com;顾菊林,E-mail:wujgjl@126.com E-mail:liaowanqing@sohu.com;wujgjl@126.com
  • 作者简介:孟云芳,女(汉族),博士研究生在读.E-mail:mengyunfang6.12@163.com;法振宗,男(汉族),博士研究生在读.E-mail:fazhenzong@163.com
  • 基金资助:

    国家973项目 (2013CB531601,2013CB531604);国家自然科学基金 (81271799,31170139);上海市医学真菌分子生物学实验室基金 (14DZ2272900)

Construction and application of blood-brain-barrier model in vitro in cryptococcal infection

MENG Yun-fang, FA Zhen-zong, FANG Wei, ZHOU Zhao-jing, YI Jiu, GU Ju-lin, LIAO Wan-qing   

  1. Shanghai Key Laboratory of Molecular Medical Mycology, Shanghai Institute of Medical Mycology, Department of Dermatology and Venereology, Changzheng Hospital, Shanghai 200003, China
  • Received:2015-01-22 Online:2015-04-28 Published:2015-04-28

摘要:

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。

关键词: 新生隐球菌, 血脑屏障, 体外模型, 致病机制

Abstract:

Objective To establish an in vitro Blood-Brain-Barrier (BBB) model,which was then applied to evaluate the capacities 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.Serotype,urease,and capsule was essential for C.neoformans to cross the Blood-Brain-Barrier.

Key words: Cryptococcus neoformans, Blood-Brain-Barrier, in vitro model, pathogenesis

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