新生隐球菌胞外蛋白水解酶对脑微血管内皮细胞的作用

Effect of extracellular proteinase secreted by Cryptococcous neoformans on brain microvascular endothelial cell

目的初步探讨新生隐球菌分泌的胞外蛋白水解酶在新生隐球菌穿越血脑屏障致病过程中的作用。方法在含有成熟的脑微血管内皮细胞的培养皿中,分别加入胞外蛋白水解酶相关成分及其特异性抑制剂后,利用相差显微镜动态观察微血管内皮细胞形态学的改变;应用免疫组织细胞化学技术检测基质金属蛋白酶-9(MMP-9)、微管相关蛋白(Tau-LRP)和低密度脂蛋白受体相关蛋白(LDL-LRP)表达的变化。结果①加入丝氨酸蛋白酶1h后可观察到内皮细胞开始收缩,面积变小,细胞间隙增宽,细胞收缩有时间依从性,至10h时仅为处理前的20%;加入丝氨酸蛋白酶+抑肽酶后细胞形态学无明显变化(P>0.05)。②加入隐球菌浓缩上清液1h后内皮细胞开始收缩,至6h时为原来的20%;加入菌株浓缩上清液+抑肽酶后细胞形态学无明显变化(P>0.05)。③丝氨酸蛋白酶使内皮细胞的MMP-9、Tau-LRP、LDL-LRP的表达上调,与对照组比较,有显著统计学差异(P<0.01)。结论新生隐球菌分泌的胞外蛋白水解酶可能通过上调MMP-9和(或)Tau-LRP、LDL-LRP的表达,诱导内皮细胞基质降解和细胞自身微管结构及紧密连接发生变化,最终导致血脑屏障通透性增加,菌体细胞穿越血脑屏障而致病。

Objective To explore the role of extracellular proteinase secreted by C. neoformans in the pathogenesis of C. neoformans penetrating the Blood-Brain-Barrier（BBB）. Methods After adding extracellular proteinase correlate formulation and treated with specific inhibitor into contain maturity brain microvascule endothelial cells cultured plate, respectively. The changes in the morphology of endothelial cell were dynmatically observed with phase-contrast light microscopy and the expression of MMP-9 ,Tau-LRP and LDL-LRP were measured with immunohistochemical method. Results BMEC began to contract after adding serine protease at lh, resulting in increased intercellular spaces and reduced cell areas. Cell contraction was time-dependent, till 10h the area was only 20% of that before treatment BMEC. After adding serine protease combine aprotinin group, un-marked change the morphology of BMEC（ P 〉 0.05 ）. BMEC began to contract after adding strain conc-supernate at lh, the area of cell was only 20% of the original BMEC after 6h. After adding strain conc-supernate combine aprotinin group, un-marked change the morphology of BMEC （ P 〉 0.05 ）. The expression of MMP-9 and/or Tau-LRP , LDL-LRP were markedly increased compared with control group （ P 〈 0.01 ）. After added inhibitor groups, the morphology and MMP-9 or Tau-LRP or LDL-LRP expression of cells had no significant difference compared with control group （ P 〉 0.05 ）. Conclusion Extracellular proteinase secreted by natural C. neoformans might up-regulate MMP-9 and/or Tau-LRP , LDL-LRP expression, induce BMEC degradation and/or changed micro-tubular structure and tight junction of itself , increase BBB permeability at the final, C. neoforman pathopoiesis through penetrating BBB.