机械牵张预处理对内毒素诱发人肺泡Ⅱ型上皮细胞损伤的影响

Effects of mechanical stretch preconditioning on endotoxin-induced damage to human type Ⅱ alveo-lar epithelial cells

目的评价机械牵张预处理对内毒素诱发人肺泡Ⅱ型上皮细胞损伤的影响。方法人肺泡Ⅱ型上皮细胞株A549细胞，长至对数生长期时，以0．5×10^6个／ml密度接种于培养板，2ml／TL，96个培养孔，采用随机数字表法，将其分为3组：正常对照组（C组，n=12）、脂多糖组（LPS组，n=36）和5％CS＋LPS组（n=48），5％CS＋LPS组分4个亚组，采用5％应变率的机械牵张分别牵张15、30、60、120min，机械牵张停止后加入LPS，LPS终浓度为1μg／ml。LPS组于LPS孵育2、4、6h时进行指标的测定，5％CS＋LPS组于LPS孵育4h时进行指标的测定，检测细胞凋亡和纤维状肌动蛋白（F—actin）表达。结果与c组比较，LPS组和5％CS＋LPS组早期细胞凋亡率和晚期细胞凋亡率升高（P〈0．05）；与LPS组孵育4h时比较，5％CS＋LPS组早期细胞凋亡率和晚期细胞凋亡率均降低（P〈0．05），F—actin重构程度明显减轻。结论5％应变率的机械牵张预处理（15—120min）可减轻内毒素诱发人肺泡Ⅱ型上皮细胞损伤。

Objective To evaluate the effects of mechanical stretch preconditioning on endotoxin- induced damage to human type Ⅱ alveolar epithelial cells. Methods Human type II alveolar epithelial cell line A549 cells at the logarithmic growth phase were seeded in 96-well plates at a density of 0. 5×10^6 cells/ml （2 ml/well）. The cells were randomly divided into 3 groups： control group （group C, n = 12） ; lipopolysaeeharide （LPS） group （n =36） and 5% CS ＋ LPS group （n = 48）. The 5% CS ＋ LPS group was further divided into 4 subgroups, and A549 cells were exposed to 5% cyclic stretch for 15, 30, 60 and 120 min in the 4 subgroups, respectively. LPS with the final concentration of 1μg/ml was added after the end of mechanical stretch in the 4 subgroups, and A549 cells were incubated for 4 h. The apoptosis in cells was detected using flow eytometry and apoptosis rate was calculated. F-aetin expression was determined using immunofluoreseence. Results Compared with group C, the early and late apoptosis rates were significantly increased in LPS and 5%CS＋ LPS groups. Compared with the value at 4 h of LPS incubation in group LPS, the early and late apoptosis rates were significantly decreased, and the reconstruction of F-actin was reduced in 5%CS ＋ LPS group. Conclusion Preconditioning with 5% cyclic stretch （15-120 rain） can mitigate endotoxin-induced damage to human type Ⅱ alveolar epithelial cells.