中文摘要：

目的观察创伤性急性肺损伤（ALI）兔血清对肺微血管内皮细胞连接蛋白40（Cx40）表达和间隙连接通道（GJC）功能的影响，及后者与内皮细胞单层通透性的关系。方法组织块贴壁法分离、培养兔肺微血管内皮细胞。细胞分为3组，对照组、创伤血清处理组和阻滞剂组。在阻滞剂组，细胞GJC阻滞剂1-庚醇（0．5mmol／L）加入到培养液中。1h后，阻滞剂组和创伤血清组的20％胎牛血清的培养液均被更换为含有20％创伤血清的培养液。对照组则只更换培养液。3组细胞继续孵育6h后，进入实验观察。分别行划痕实验、Cx40蛋白免疫印迹、单层细胞通透性和细胞内Ca^2±钙浓度检测。结果Cx40蛋白印迹见创伤血清组与阻滞剂组蛋白表达水平较对照组有明显降低，且2组之间亦不同，阻滞剂组蛋白水平较创伤血清组低，吸光度和值减少（对照组726293．2±83684．1，创伤血清组397798．1±87145．7，阻滞剂组316977．6±76325．9，n=4，P〈0．05）。3组细胞划痕边缘的细胞均被染料荧光黄（LY）染色，LY在对照组细胞扩散得最远，显色的周边细胞的数量最多，创伤血清组次之，阻滞剂组显色的细胞数量最少，相邻细胞与边缘细胞比值的差异有统计学意义（23±5、11±3比7±2，n=4，P〈0．05）。3组细胞伊文思蓝清除率均随着时间的延长而增加，且3组细胞间亦有不同，阻滞剂组清除率高于创伤血清组和对照组。组间差异和时间点差异均有统计学意义（P〈0．05）。通过共聚焦显微镜的观察，3组细胞荧光强度不相同，阻滞剂组荧光最强，对照组最弱。创伤血清组和阻滞剂组细胞内Ca^2±浓度均显著高于对照组[（494．80±41．94）、（569．80±6．70）nmol／L比（158．80±13．09）nmol／L，P〈0．05]。结论创伤性ALI动物血清抑制肺微血管内皮细胞Cx40表达，进而引起GJc功能下降，导致肺血管内皮?

英文摘要：

Objective To investigate the influence of traumatic acute lung injury （ALI） rabbit serum on gap junction channel （ GJC ） function and connexin40 （ Cx40 ） expression, and the correlation of GJC and Cx40 with rabbit pulmonary microvascular endothelial cells permeability. Methods Cultured pulmonary microvcssel endothelial cells were divided into three groups, control （G 1）, injured serum （ G ） , and blocker agent （Gblocker）. GJC function was assessed by scrape-loading and dye transfer techniques. Pulmonary microvessel endothelial cells permeability was measured by Evans blue-labeled albumin transfer, and the expression of Cx40 was measured by Western blotting. Intracellular free calcium concen- tration was measured by fluo-3am. Results Injured serum decreased GJC function and the level of Cx40, which was aggravated by the GJC blocker （ control 726 293.2 ＋ 83 684. 1, serum 397 798.1 ＋ 87 145.7, blocker 316 977. 6 ＋ 76 325.9, n = 4, P 〈 0. 05 ）. Similarly, pulmonary microvascular endothelial cells permeability was increased significantly in G and Gblocker as compared with Gcont, oI （ P 〈 0.05 ）. After treat- ment with injured serum in combination with gap junction blocker in vitro, intracellular free calcium con- centration was increased [ control （494. 80 ＋ 41.94） nmol/L, serum （569.80 ＋ 6. 70） nmol/L, blocker （ 158. 80 ＋ 13.09） nmol/L, P 〈 0.05 ]. Conclusion Traumatic ALI rabbit serum inhibits the Cx40 expression and down-regulates GJC function, which contributes to the increase of pulmonary microvascular endothelial cells permeability after ALI. This mechanism may participate in the increase of pulmonary vascular permeability and promote the morbidity of ALI after trauma.