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目的观察低氧环境对人血管内皮细胞血管紧张素转换酶2(ACE2)表达的影响,并初步探讨其作用机制。方法人脐静脉内皮细胞株(HUVECs),进行贴壁细胞培养;采用免疫组织化学方法对细胞株进行Ⅷ因子鉴定;设立低氧实验组与常氧对照组。低氧组条件为1%O2+94%N2+5%CO2,常氧对照组的细胞置于5%CO2+95%空气孵箱内培养,37℃培养3h、6h、12h、24h,收集培养液和细胞;采用免疫组化鉴定ACE2蛋白合成情况,实时荧光定量RT-PCR检测ACE2mRNA表达水平。结果①与常氧组对照,低氧3h、6h、12h、24h组ACE2蛋白表达均有所下降(P均≤0.00625),其中低氧3h组的阳性率最低为14.53%,并且随时间延长呈现逐渐上升趋势;②与常氧对照组相比,低氧3h、6h、12h、24h组ACE2mRNA表达量均有所下降,其中低氧3h组ACE2mRNA相对表达率为0.281(P=0.031),低氧6h组为0.445(P=0.034),低氧12h组为0.794(P=0.434),低氧24h组为0.891(P=0.692)。结论在24h内,低氧使人血管内皮细胞ACE2蛋白的合成以及ACE2mRNA的表达减少,并且以3h最为明显,后出现逐渐升高趋势。这种先减少后增加的趋势,提示逐渐升高的ACE2对血管内皮缺氧损伤后的肾素血管紧张素系统(RAS)的反向调节有着重要作用。
Objective To investigate the effect of hypoxic environment on the expression of angiotensin-converting enzyme 2 (ACE2) in human vascular endothelial cells and to explore its mechanism. Methods Human umbilical vein endothelial cells (HUVECs) were cultured in adherent cells. Immunohistochemistry was used to identify the cell lines for factor Ⅷ. Hypoxic and normoxic control groups were established. The conditions of hypoxia group were 1% O2 + 94% N2 + 5% CO2. The cells in normoxic control group were cultured in 5% CO2 + 95% air incubator at 37 ℃ for 3h, 6h, 12h, 24h The expression of ACE2 protein was detected by immunohistochemistry. The expression of ACE2 mRNA was detected by real-time fluorescence quantitative RT-PCR. Results ① Compared with normoxia group, the expression of ACE2 protein in hypoxia 3h, 6h, 12h and 24h groups decreased (P <0.05), and the lowest positive rate in hypoxia 3h group was 14.53% (2) ACE2mRNA expression decreased at 3h, 6h, 12h and 24h after hypoxia compared with normoxia control group. The relative expression rate of ACE2mRNA in hypoxia 3h group was 0.281 (P = 0.031), hypoxia 0.445 in 6h group (P = 0.034), 0.794 in 12h hypoxia group (P = 0.434), and 0.891 in hypoxia 24h group (P = 0.692). Conclusions Within 24h, hypoxia reduced the synthesis of ACE2 protein and the expression of ACE2 mRNA in human vascular endothelial cells, and became the most obvious after 3 hours, then increased gradually. This trend of first decreasing and then increasing suggests that increasing ACE2 may play an important role in the reverse regulation of the renin-angiotensin system (RAS) following endothelial hypoxia.