新生隐球菌通过S100A10活化尿激酶-纤溶酶系统侵袭血脑屏障的机制研究

Cryptococcus traversal across the Blood-Brain Barrier though S100A10-dependent activation of urokinaseplasmin system

目的验证隐球菌可以上调脑微血管内皮细胞S100A10基因来活化尿激酶-纤溶酶系统,从而更易穿过血脑屏障。方法 1将小鼠脑微血管内皮细胞bEnd.3和新生隐球菌B3501共孵育,检测加菌的实验组和不加菌对照组S100A10、UPA基因和蛋白的表达量。2用慢病毒感染血管内皮细胞,比较S100A10基因正常组和沉默组的S100A10和UPA基因和蛋白的表达量。3构建Transwell血脑屏障模型,比较S100A10基因正常组和沉默组隐球菌通过的差异性,用底物显色法检测尿激酶-纤溶酶系统的相关活化情况。结果 1新生隐球菌B3501和bEnd.3共孵育,Real-time PCR显示S100A10和UPA的mRNA高表达;Western-blot显示S100A10目的蛋白和UPA蛋白高表达。2沉默S100A10基因后,S100A10和UPA基因及蛋白表达分别受到抑制。3隐球菌添加到Transwell体外血脑屏障模型中,S100A10基因未沉默组通过血脑屏障的隐球菌数量比S100A10基因沉默组多,尤其是8h、16h、24h,两组差异有统计学意义,P<0.05;未沉默S100A10基因的对照组的纤溶酶原和尿激酶的活化程度高于沉默S100A10基因的实验组,其中纤溶酶原在8h、16h、24h,尿激酶在16h、24h,两组差异有统计学意义,P<0.05。结论隐球菌能同时使内皮细胞S100A10基因、UPA基因的高表达,活化尿激酶-纤溶酶系统,提高其通过血脑屏障的能力。

Objective To find if Cryptococcus traversal across the blood-brain barrier though SlOOAlO-dependent activation of urokinase-plasmin system.Methods @The mouse brain microvascular endothelial cells bEnd.3 and Cryptococcus neofor- marts B3501 were co-incubated.Then the expression of S100A10 gene and UPA genewere testedExpressionof S100AI0 and UPA in S100A10 normal and silent groups of endothelial cellsInfected with lentivirus were also compared.The abilities of Cryptococcus S100A10 normal and silent groups traversal across the transwell blood-brain barrier mode! were eompared.Uro- kinase-related activation of plasminogen was tested by the chromogenic substrate assay.Results In bEnd.3 incubated with Cryptococcus neoformans B3501,Real-time PCR display high expression of mRNA of S100A10 and UPA;Western-blot show S100A10 protein and UPA protein overexpression.While S100A10 gene been silenced,S100A10 and UPA xpressions were suppressed.@Cryptococcal traversal across the transwell in vitro BBB model in S100A10 silenced group was less than that of controlgroup.The diffenees on 8 h, 16 h,24 h were especially statistically significant,P〈0.05.The activation of urokinase and plasmin was also lower than that of control group. The diffences of plasminogen activation on 8 h, t6 hand 24 h in two groups,the diffences of urokinase activation on 16 h and 24 h in two groups,were statistically significamrespeetively.Conclu- sion Cryptoeoccus traversal across the bl＇ood-brain barrier might be though S100A10-dependent activation of urokinase-plas- min system.In the meantime, the S100A10 gene and UPA gene of brain microvascular endothelial cells were highly expressed.