间歇低氧致人脐静脉内皮细胞凋亡与线粒体细胞色素C释放的相关性

Correlation between intermittent hypoxia-induced apoptosis of human umbilical vein endothelial cells and release of cytochrome C from mitochondrla

目的探讨间歇低氧（IH）致人脐静脉内皮细胞（HUVECs）凋亡与线粒体细胞色素C（cytC）释放的相关性。方法 研究分为IH组和对照组，IH组HUVECs接受IH刺激，置于1%O2 5 min与21% O2 5 min循环交替（37 ℃，5% CO2）的孵育箱，观察时间分别为8、16、24 h；对照组细胞则置于普通常氧孵育箱（37 ℃，5% CO2）内24 h。应用Annexin V－FITC/PI检测细胞凋亡率，电镜观察细胞线粒体结构变化，差速离心法分离HUVECs胞质与线粒体并分别检测各组分中cytC的相对表达量。分析cytC的释放与细胞凋亡的相关性。结果 IH组HUVECs内线粒体结构发生明显变化，出现线粒体肿胀、嵴断裂、空泡化等现象；而对照组线粒体超微结构仍保持完整。IH组8、16、24 h的HUVECs凋亡率分别为（6.710 ± 0.599）%、（8.863 ± 1.190）%、（9.607 ± 1.266）%，均显著高于对照组的（2.450 ± 0.795）%（均P〈0.05）；且凋亡率随IH处理时间延长而增加。胞质cytC含量与细胞早期凋亡率呈正相关（r＝0.741, P＝0.022）。结论 IH可致内皮细胞线粒体cytC释放入胞质，促进细胞凋亡，参与IH致血管内皮损伤。

ObjectiveTo explore the correlation between intermittent hypoxia （IH）-induced apoptosis of human umbilical vein endothelial cells （HUVECs） and release of cytochrome C （cytC） from mitochondria.MethodsHUVECs in IH groups were exposed to repetitive cycles （1% O2 5 min alternating with 21% O2 5 min, 37 ℃, 5% CO2） for different exposure time （8, 16 and 24 h, respectively）, while the control group were placed in an ordinary incubator （21% O2, 37 ℃, 5% CO2） for 24 h. Apoptosis rate of HUVECs were determined by using Annexin V-FITC/PI apoptosis detection kit. The mitochondria ultrastructure was observed under transmission electron microscope （TEM）. The mitochondria and cytoplasm of HUVECs were isolated by differential centrifugation, and the expression cytC in two separations were detected by Western blot. The correlation between the release of cytC and apoptosis rate was analyzed.ResultsTEM showed that mitochondria in HUVECs treated with IH appeared significant swelling, cristae rupture and vacuolization, compared with intact mitochondria in control group. Apoptosis rates in IH groups of 8, 16 and 24 h were significantly higher than control group[（6.710±0.599）%, （8.863±1.190）% and （9.607±1.266）% vs （2.450±0.795）%]in a time-dependent manner （all P〈0.05）. Apoptosis rates in IH groups positively correlated with cytC expression in cytoplasm （r=0.741, P=0.022）.ConclusionIH-induced cytC release from mitochondria promotes apoptosis of HUVECs, which participates in IH-induced endothelial injury.