缺氧对大鼠肺泡上皮细胞凋亡及缺氧诱导因子-1α表达的影响

目的 探讨氯化钴(cobalt chloride,CoCl2)化学缺氧对大鼠肺泡上皮细胞(alveolar epithelial cell,AEC)凋亡的影响及相关凋亡蛋白缺氧诱导因子-1α(hypoxia inducible factor-talpha,HIF-1α)的表达变化及意义.方法 将AEC进行体外培养,分4组,每组6个样本.缺氧4 h组:选用化学缺氧物质CoCl2 500/μmol/L为作用浓度,诱导AEC缺氧,共培养4 h;缺氧24 h组:与CoCl2共培养24 h;缺氧48 h组:与CoCl2共培养48 h;常氧对照组:不做任何特殊处理.利用荧光显微镜及流式细胞仪检测细胞凋亡率,透射电子显微镜进行细胞凋亡的形态学观察,Western印记法检测HIF-1α蛋白的表达情况.结果 (1)荧光显微镜的结果 显示缺氧4、24、48 h组细胞凋亡率分别为(5.83±0.76)%、(15.00±3.28)%和(51.50±3.00)%,均高于常氧对照组(1.50±0.50)%](P＜0.05);(2)流式细胞仪的结果 与荧光显微镜的结果一致;(3)透射电子显微镜可以观察到AEC凋亡的形态学改变,缺氧24 h显著;(4)缺氧4、24、48 h组HIF-1α蛋白的表达分别为0.69±0.035、1.02±0.044和0.71±0.046,均高于常氧对照组(0.21±0.026)(P＜0.01);(5)各组HIF-1α蛋白变化与荧光显微镜及流式细胞仪检测的细胞凋亡率变化均呈正相关(r=0.484,P=0.016;r=0.713,P=0.009).结论 缺氧对大鼠AEC的生存有抑制作用,这种抑制作用与缺氧引起的细胞凋亡有关;HIF-1α可能参与了缺氧诱导AEC凋亡的发生,而AEC凋亡可能又参与了缺氧肺损伤的发病.

Significance of hypoxia induced alveolar epithelial cell apoptosis and hypoxia inducible factor-1alpha expression

Objective To investigate the effects of cobalt chloride(COCl2)induced rat alveolar epithelial cell apoptosis and the significance of related apoptosis protein hypoxia inducible factor-lalpha(HIF-1 a)expression. Methods Alveolar epithelial cells were cultured in vitro and divided into four groups with 6 samples in each.Hypoxia 4 h group:added 500μmol/L COCl2 for 4 hours inducing alveolar epithelial cell hypoxia;hypoxia 24 h group:co-cultured with COCl2 for 24 hours;hypoxia 48 h group:co-cultured with CoCl2 for 48 hours;control group:no CoCl2 added.The apoptosis rates were detected by fluorescent microscope and flow cytometry.The apoptosis morphological changes of the cell were observed by transmission electron microscopy.The protein expression of HIF-la was detected bv Western Blot. Results (1)Fluorescent microscope results showed that apoptosis rate in hypoxia 4 h、24 h、48 h group(5.83±0.76)%,(15.00±3.28)%and(51.50±3.00)%],were higher than control group(1.50±0.50)%](P＜0.05).(2)Flow cytometry analysis reached the same results as with fluorescent microscope.(3)Typical morphological changes of apoptosis were observed in hypoxia 24 h group.(4)HIF-1α expression in hypoxia 4 h、24 h and 48 h groups0.69±0.035,1.02±0.044 and 0.71±0.046],were higher than control group(0.21±0.026)(P＜0.01).(5)Positive correlations were found between relative amount of HIF-1αprotein and apoptosis rate by fluorescent microscope and flow cytometry analysis(r=0.484,P=0.016;r=0.713,P=0.009).Conclusions Hypoxia could induce apoptosis of alveolar epithelial cells,which may participate in the pathogenesis of hypoxia-induced lung injury.