内毒素对肺微血管内皮细胞形态学及结构损伤的研究

Studies on the Pulmonary Microvascular Endothelial Cells Morphology and Structure Injury Caused by Endotoxin

目的建立大鼠肺微血管内皮细胞(PMVECs)系并探讨脂多糖(LPS)对PMVEC形态学及细胞结构的作用。方法组织块贴壁法培养原代大鼠PMVEC并进行综合鉴定,对各代细胞进行形态学观察。观察正常组、LPS(高浓度)4h组、LPS8h组、LPS(高浓度)12h组细胞形态的变化,利用PI染色法观察细胞核的变化,并利用透射电镜观察正常组、LPS(低浓度)24h组细胞内亚结构的改变。结果培养的肺微血管内皮细胞系中,各代细胞形态学有差异,同时原代细胞与传代细胞的生长速度也存在差异。高浓度LPS导致细胞形态发生变化呈时间依赖性:LPS作用4h后,部分细胞开始从多角形变成圆形;LPS作用8h后,细胞膜出现皱褶,边界不清,细胞浆内密度不均,细胞核变化不大;LPS作用12h后,大部分细胞形态呈圆形,细胞膜丧失完整性,胞浆和胞核难以区分,细胞核发生皱缩,染色质分布不均。低浓度LPS作用24h导致细胞内亚结构如线粒体、内质网发生病理性改变。结论培养的肺微血管内皮细胞系具有多态性,脂多糖诱导PMVEC形态学改变及亚结构的损伤,表明LPS相关的ARDS发病机制中,可能存在多条信号通路调控肺微血管内皮细胞功能的改变。

Objective To establish the rat pulmonary microvascular endothelial cells （PMVECs）and investigate the effects of lipopolysaecharide（LPS） on morphology and structure of PMVEC. Methods Primary rat PMVEC was cultured by tis- sue-pieces inoculation and comprehensively appraised. And the morphology of each generation was observed. Morphological changes of the cells in normal group, LPS 4h group（ of high concentrations ） , LPS 8h group and LPS 12h group （of high concentrations）were observed and the changes of nucleus was observed by using PI staining. And changes of intercellular substructure in normal group and LPS 24h group（ of low coneentrations）were observed with transmission electron microscopy. Results There were morphologi- cal differences among generations in PMVECs. And primary culture cells and subculture cells also differed in growth rates. High concentration of LPS contributed to time dependence of cellular morphological changes ：4h after LPS effected, some cells began from polygon into round;8h after LPS effected after 8 h, cell membrane folds, the boundary is not clear, density of cytoplasm became un- even and cell nucleus changed little;12h after LPS effected, most of the cells morphology became circular, cell membrane lost integ- rity,cytoplasm and nucleus were difficult to distinguish,nucleus shrunk and chromatin distributed; 24h after low concentration of LPS effected, intercellular substructure such as mitochondria and endoplasmic reticulum had pathological changes. Conclusion Cultured PMVECs are polymorphic. And LPS induces morphologic changes and substructure injury of PMVEC ,which indicates that in the LPS-related pathogenesis of ARDS, there may be multiple signaling pathways regulating changes of lung mierovascular endo- thelial cell function.