急性肺损伤炎症环境中TNF-α介导肺通透性增加的机制研究

摘要

目的 探讨急性肺损伤(ALI)时,炎症环境中肿瘤坏死因子(TNF-α)介导肺通透性增加的机制.方法 30只SPF级健康Balb/c小鼠分为6组,对照组、0 h实验组、1 h实验组、2 h实验组、4 h实验组、8 h实验组,每组5只.实验组采用腹腔注射脂多糖(LPS)法,根据体重用要求的剂量于不同时间点进行注射,对照组注入同等剂量的生理盐水.LPS诱导急性肺损伤模型后,眼眶取血制备血清,游离肺组织碾磨制备上清,ELISA检测肺组织上清液中TNF-α的含量;LPS诱导急性肺损伤模型后,尾静脉注射伊文思蓝,在620 nm波长下检测不同时间点伊文思蓝溶液的吸收值;Western Blotting检测急性肺损伤小鼠模型中肺组织的低氧诱导因子-1α(HIF-1α)和血管扩张刺激磷蛋白(VASP)的蛋白表达水平改变.结果 LPS能成功诱导急性肺损伤模型,4 h实验组和8 h实验组的ELISA结果显示,实验组肺组织上清液中的TNF-α的含量明显升高;LPS造模后,尾静脉注射伊文思蓝,在620 nm波长下检测伊文思蓝溶液的吸收值,各组1 h开始升高,4 h实验组较0 h实验组明显升高,且差异有显著性(P<0.01);在小鼠的急性肺损伤模型中,实验组小鼠肺组织中HIF-1α的蛋白表达在4 h和8 h明显增加,同时VASP的蛋白表达在4 h和8 h明显降低.结论 急性肺损伤症过程中,TNF-α可诱导HIF-1α的活化,进而引起VASP的表达下调,肺泡-毛细血管屏障损害,随着时间的增长,情况有所减退.%Objective To investigate the effects and mechanisms of TNF-α increasing the tight junction permeability in lung tissue asso-ciated with acute lung inflammation. Methods Healthy Balb/ c mice were divided into 6 groups:control group,0 h experimental group,1 h ex-perimental group,2 h experimental group,4 h experimental group,8 h experimental group,5 rats in each group. The experimental group was in-jected with abdominal cavity,injected with the required dose according to the weight,and the control group was injected with the same dose normal saline. TNF-α in the serum and lung tissue were deby ELISA in LPS-induced acute lung injury mice model;Evans blue was used to detect the permeability of pulmonary capillary. Western Blotting was used to analyze HIF-1α and VASP protien expression level during the development of acute lung injury. Results In the LPS-induced model of ALI,the experiment showed that the concentration of TNF-α increased at 1 h and at 4 h it reached peak by Elisa. Evans Blue was injected in tail vein,and then Evans Blue'absorption value was detected at 620 nm wavelength which reached its peak at 4 h. After LPS-inducing acute lung injury,HIF-1αprotein expression in the mice lung tissue increased significantly at 4 h and 8 h,while VASP protein expression decreased. Conclusion During acute pulmonary inflammation process,VASP is down regulated through TNF-α inducing HIF-1α activation,which plays an important role in the impairment of alveolar-capillary barrier.