CD44在新生隐球菌体外感染血脑屏障中对单核细胞迁移的影响

目的新生隐球菌（Cryptococcus neoformans, Cn）荚膜主要毒力因子透明质酸由CPS1基因编码，能与宿主细胞受体CD44
结合介导真菌侵袭。本文目的明确CD44分子在Cn体外感染血脑屏障模型中对单核细胞黏附人脑微血管内皮细胞（HBMEC）
并迁移穿越血脑屏障的影响。方法用单层HBMEC铺趋化小槽构建体外血脑屏障模型，利用Cn野生株B4500FO2、CPS1基因
缺失株TYCC645#32和CPS1基因回补株PCIP，分别感染模型中的HBMEC，然后在上槽液中加入人白血病单核细胞（THP-1），
计数从上槽液中迁移到下槽液中的单核细胞数量，观察Cn感染与单核细胞迁移时间和剂量关系；用抗CD44 单克隆抗体和
CD44抑制剂Bikunin分别作用于模型中单层HBMEC，通过趋化实验检查抗CD44单克隆抗体和Bikunin对Cn感染模型中单核
细胞迁移的抑制效应。结果黏附和趋化实验结果显示，Cn感染HBMEC后，相比PBS对照组，THP-1黏附率和趋化效应均明
显增加（P<0.01），且黏附率和迁移率随Cn感染量和感染时间的增加而显著上升（P<0.05）。通过抗CD44单克隆抗体和CD44
阻断剂Bikunin分别作用于Cn感染后的HBMEC，结果发现THP-1黏附率和迁移率均显著降低（P<0.01），且在一定范围内分别
随抗体（0~1 μg）和抑制剂（0~20 nmol/L）剂量的增加而减低。不同Cn菌株荚膜透明质酸的表达差异对THP-1细胞黏附和迁移
有一定影响：与野生株相比，TYCC645#32 感染组的THP-1 黏附率和迁移率均明显降低（P<0.01，P<0.05），而回补株PCIP 的
THP-1黏附率和迁移率有所增加。结论体外血脑屏障模型中人脑微血管细胞表达CD44分子可能对单核细胞黏附内皮细胞
和迁移通过血脑屏障起重要作用；荚膜透明质酸能介导Cn诱导的单核细胞黏附和迁移。

Role of CD44 in monocyte transmigration across Cryptococcus neoformans-infectedblood-brain barrier in vitro

Objective To explore the role of CD44 in monocyte adhesion to human brain microvascular endothelial cells
(HBMECs) and monocyte migration across an in vitro model of blood-brain barrier (BBB) infected by Cryptococcus neoformans
(Cn). Methods An in vitro blood-brain barrier model was constructed using a transwell chamber covered with a HBMEC
monolayer. The wild-type strain of Cn B4500FO2, TYCC645#32 strain with CPS1 gene deletion and PCIP strain with CPS1
complementation were chosen to infect the monolayer HBMECs. THP-1 cells were added to the upper chamber of transwell,
and the relative migration rate was determined by counting the number of the cells entering the lower chambers. The
inhibitory effects of anti-CD44 monoclonal antibody and the CD44 inhibitor bikunin were examined on THP-1 binding to and
migration across HBMECs. Results Cn infection of the HBMECs caused markedly enhanced THP-1 cell adhesion and
migration across the monolyers (P<0.01) dependent on Cn concentration and exposure time. Addition of anti-CD44
monoclonal antibody and bikunin significantly lowered THP-1 adhesion and migration rates in the BBB model with
Cn-infected HBMECs (P<0.01) with a dose dependence of the antibody (within 0-1 μg) and inhibitor (within 0-20 nmol/L). Both
THP-1 adhesion rate and migration rate were lowered in the BBB model infected with CPS1 gene-deleted Cn but increased in
the model infected with the complemented strain compared with those in the wild-type strain-infected model. Conclusion In
the in vitro BBB model, CD44 expressed on HBMECs may play an essential role in monocyte adhesion to and migration across
the BBB. The capsular hyaluronic acid may mediate Cn-induced monocyte adhesion and migration.