肿瘤坏死因子α对豚鼠耳蜗血管纹微血管内皮细胞通透性的影响

摘要

Objective To perform an initial study on possible molecular regulatory mechanisms of TNF-α to the permeability of strial capillary endothelial cells in guinea pig cochlea. Methods Strial capillary endothelial cells in guinea pig cochlea was dissociated and cuhureed to establish a model for its permeability in vitro.,The animals were divided into TNF-α,TNF-α+L-arginine (L-Arg),TNF-α +NG-minomethyl-L-arginine(L-NMMA) and control groups.In order to measure the altemations of permeation rate,Evans blue was used in different time by different concentrations to detect contents of F-actin in each group under immunofluorescence laser scanning confocal micrescope.Results TNF-α increased permeability of Blrial capillary endothelial cells notablely,and permeation rate to Evans blue heightened siguificandy (P＜0.01) and increased under higher concentrations of TNF-α as well as lengthened the durations.Being as a NO donator,L-Arg siguitlcanfly enhanced the ability of TNF-α which increasing permeability of strial capillary cndothelial cells;permeation rate to Evans blue beightened significantly(P＜0.01),which obviously rose up when the concentration of Evans blue added.L-NMMA,NO inhibitor,was able to weaken the ability of TNF-α which increasing permeability of strial capillary endothelial cells:permeation rate to Evans blue decreased siguificantly (P＜0.01),which obviously fell down when the concentration of L-NMMA added.TNF-α decreased F-actin in strial capillary endothelisl cells(P＜0.01).more TNF-α, less F-actin;L-Arg could be further the inhibition to F-actin content(P＜0.05);L-NMMA held back the trend(P＜0.05).Conclusions TNF-α can increase permeability of strial capillary endothelial cells of suinca pig cochlea.NO may be one of the important molecular regulators to permeability of strial capillary endothelial cells by TNF-α,and changes of F-actin content probably relate to the regulation in these cells.%目的 探讨肿瘤坏死因子α(TNF-α)对豚鼠耳蜗血管纹微血管内皮细胞通透性的影响及可能的分子调控机理.方法 体外分离培养豚鼠耳蜗血管纹微血管内皮细胞并建立内皮细胞通透性体外模型,观察TNF-α、TNF-α连同L-精氨酸(L-arginine,L-Arg)或L-单甲基精氨酸(NG-monomethyl-L-arginine,L-NMMA)对内皮细胞通透性的影响.按照作用时间和浓度的不同,分别检测内皮细胞对伊文思蓝通透率的变化,并通过免疫荧光激光共聚焦扫描显微镜测定各组标本中的F-肌动蛋白(F-actin)含量的变化.结果 TNF-α能显著增加耳蜗血管纹微血管内皮细胞的通透性,使伊文思蓝的通透率升高(P值均＜0.01),且随TNF-α浓度的提升和作用时间的延长而增加.一氧化氮(nitric oxide,NO)供体L-Arg能增加TNF-α作用下耳蜗血管纹微血管内皮细胞的通透性,使伊文思蓝的通透率升高,差异具有统计学意义(P值均＜0.01),且随着L-Arg浓度的增加内皮细胞对伊文思蓝的通透率也明显增加.一氧化氮合酶(nitric oxide synthase,NOS)抑制剂L-NMMA可降低TNF-α作用下耳蜗血管纹微血管内皮细胞的通透性,使伊文思蓝的通透率下降,差异具有统计学意义(P值均＜0.01),且随着L-NMMA浓度的增加内皮细胞对伊文思蓝的通透率也明显下降.TNF-α能使内皮细胞中的F-actin含量明显减少(P＜0.01),且TNF-α的浓度越高F-acitn的含量降低越明显;L-Arg能进一步降低F-actin的含量(P＜0.05);而L-NMMA则能抑制这种趋势(P＜0.05).结论 TNF-α能增加耳蜗血管纹微血管内皮细胞的通透性,NO可能是TNF-α调控内皮细胞通透性的重要分子机制之一;耳蜗微血管内皮细胞中F-acfin含量的改变可能与其通透性的调控有关.