伪狂犬病病毒感染小鼠基质金属蛋白酶-9介导紧密连接蛋白损伤血脑屏障的研究

旨在探究伪狂犬病病毒(pseudorabies virus, PRV)对小鼠血脑屏障(blood-brain barrier, BBB)通透性的影响及基质金属蛋白酶-9(matrix metalloproteinase-9,MMP-9)与紧密连接(tight junction, TJ)的相关性。将昆明SPF小鼠96只,随机平均分为对照组和3个试验组,24只对照组小鼠滴鼻生理盐水,72只试验组小鼠滴鼻感染PRV,将试验组分为感染24 h组、感染48 h组和感染72 h组,每组24只。观察临床症状并采用H&E染色观察病理损伤情况;通过改良型神经学评分和脑组织病毒载量测定评价PRV感染后神经损伤情况与病毒载量的关系;通过干湿重法和伊文斯蓝染色法探究血脑屏障的通透性;通过mRNA和蛋白表达差异水平探究MMP-9和TJ的相关性。结果显示,攻毒后脑组织病变程度随时间延长而加剧,在攻毒48 h后,脑组织病毒载量出现显著性差异;神经功能损伤评分在第72小时达到最高;干/湿重值及脑组织伊文斯蓝浓度呈时间依赖性;MMP-9与TJ的相关性结果表明,PRV攻毒后MMP-9表达量增加,TJ表达量减少。结果表明,PRV感染通过MMP-9介导的TJ蛋白表达量的降低损害BBB的完整性及通透性。综上所述,PRV感染可能通过MMP-9介导的破坏来损害BBB,为进一步阐明PRV入侵机制奠定基础。

Effects of Simulated Weightlessness on Tight Junction Protein Occludin and Zonula Occluden-1 Expression Levels in the Intestinal Mucosa of Rats

This study investigated the tight junction（TJ） protein expression of the intestinal mucosa in a rat tail-suspension model under simulated weightlessness.Twenty-four Wistar rats were randomly divided into three groups：CON group（n=8）,control; SUS-14 d group（n=8）,tail-suspension for 14 days; SUS-21 d group（n=8）,tail-suspension for 21 days.Occludin and Zonula Occluden-1（ZO-1） expression levels were determined by immunohistochemical analysis and mRNA fluorescent quantitative PCR.Plasma levels of diamine oxidase（DAO） and d-lactate were determined using enzymatic spectrophotometry.Immunohistochemical results for occludin and ZO-1 showed disruption of the TJs in the intestinal mucosa in SUS-14 d and SUS-21 d groups.The expression levels of occludin and ZO-1 in SUS-21 d group were lower than those in SUS-14 d group,and significantly lower than those in CON group（Occldin：0.86±0.02 vs 1.01±0.03 vs 1.63±0.03 and ZO-1：0.82±0.01 vs 1.00±0.02 vs 1.55±0.01,P〈0.01）.Moreover,the levels of plasma DAO and d-lactate in SUS-21 d group were higher than those in SUS-14 d group,and significantly higher than those in CON group（DAO：27.58±0.49 vs 20.74±0.49 vs 12.94±0.21 and d-lactate：37.86±0.74 vs 28.26±1.01 vs 17.76±0.91,P〈0.01）.There were significant negative correlations between occludin or ZO-1 expression levels and DAO（r2=0.9014,r2=0.9355,P〈0.01） or d-lactate levels（r2=0.8989,r2=0.9331,P〈0.01）.Occludin and Zo-1 were reduced in intestinal mucosa both in mRNA and protein levels in the rat tail-suspension model.The significant negative correlations between expression levels of TJs and plasma levels of DAO or d-lactate support the hypothesis that intestinal permeability is increased due to a decrease in TJ protein expression during tail-suspension from 14 days to 21 days.

鼻脑递送:葛根素在嗅神经鞘细胞的摄取及在Calu-3细胞的转运研究（英文）

目的:建立鼻腔给药直接通路与间接通路体外模型,考察微乳对葛根素在嗅神经鞘细胞(OECs)的摄取和在Calu-3细胞单层的转运影响并探讨作用机制。方法:原代培养OECs,传代培养Calu-3细胞分别模拟穿细胞途径直接通路和细胞旁路途径间接通路。通过MTT实验考察葛根素溶液和微乳的细胞毒性。分别考察葛根素在Calu-3细胞单层的转运和OECs中的摄取。免疫组化考察微乳对Calu-3单层的紧密连接蛋白影响。结果:原代培养嗅神经鞘细胞并通过差速贴壁和阿糖胞苷纯化,得到的纯化细胞可作为体外嗅神经通路模型。葛根素溶液在Calu-3细胞模型的转运PAPP值为(1.19±0.11)(A→B)和(1.21±0.06)(B→A)。葛根素微乳的转运PAPP值显著高于葛根素溶液,PAPP值为(1.64±0.08)(A→B)和(1.86±0.17)(B→A)。OECs对溶液中葛根素的摄取在30min达到平衡,浓度为0.095mg/g,对微乳中葛根素的摄取60min达到平衡,浓度为0.352mg/g,显著高于葛根素溶液组。结论:本实验建立的Calu-3和OECs细胞模型可以在体外分别模拟鼻脑递送直接通路和间接通路,微乳可促进葛根素在体外鼻脑递送模型的转运,其作用机制与打开Calu-3细胞间紧密连接,增加葛根素细胞旁路转运。