急性肺损伤时间隙连接通道调控肺血管通透性的机制研究

目的探讨间隙连接通道(GJC)是否通过调节胞内钙离子浓度,进而调控肺血管通透性,最终导致急性肺损伤的发病。方法应用小口径步枪致伤制造急性肺损伤动物模型,检测肺含水量、伊文思蓝漏出率,并应用免疫组织化学方法检测肺血管内皮Cx40表达;培养肺微血管内皮细胞,分别给予达氏修正伊氏培养基(DMEM)、伤后动物血清和GJC通道阻滞剂,应用染料划痕实验检测GJC功能、伊文思蓝漏出实验检测单层内皮细胞通透性、Fluo-3AM钙离子荧光探针检测胞内钙离子浓度。结果动物实验中,伤后Cx40表达随时间延长逐渐降低,肺血管通透性则呈递增趋势,二者呈负相关(r=-0.934,P<0.05)。离体实验中,伤后动物血清降低GJC功能,Cx40表达降低,当应用通道阻断剂后,GJC功能和Cx40表达降低程度更甚;此时肺微血管内皮细胞单层通透性增加,胞内钙离子浓度增加,应用GJC通道阻断剂后,这种效应更加明显。结论肺血管内皮细胞GJC对肺血管通透性有调节作用,胸部枪弹伤后,肺血管内皮Cx40表达降低,引起GJC功能下降,导致胞内钙超载,最终导致肺血管通透性增加和急性肺损伤的发病。

Research of the mechanism of gap junction channels modulating pulmonary vaso-permeability in acute lung injury

Objective To study the mechanism of gap junction channels(GJCs)modulating pulmonary vaso-permeability in acute lung injury(ALI).Methods Firstly we used animal model of ALI and correlated Cx40 by inmunohistochemistry in lung with Evans′Blue leak.Then cultured pulmonary microvessel endothelial cells were divided into three groups:Gcontrol,Gserumand Gblocker.Injured serum was obtained from ALI animals.Initially Gblockerwas treated with the blocker of GJCs,and then Gserumand Gblockerwere stimulated respectively with the injured serum.GJCs,the permeability of cells monolayer and intracellular Ca2+were measured.Results Cx40level time-dependently decreased,whereas Evans′Blue leak increased.Cx40level and Evans′Blue leak exhibited a strong inverse correlation(r=-0.934,P<0.05).Injured serum decreased GJCs and expression of Cx40,the blocker aggravated this effect.Similarly,when pulmonary microvessel endothelial cells monolayer was treated with injured serum,the permeability and intracellular Ca2+both increased.These effects were also aggravated with the blocker.Conclusion Depression of GJCs of pulmonary microvessel endothelial cells increased pulmonary vaso-permeability in ALI.This effect may be mediated by the overload of intracellular calcium.