白藜三醇促进缺氧条件下人脐静脉内皮细胞的血管生成作用  

Effect of Resveratrol for Improving Angiogenesis in Human Umbilical Vein Endothelial Cell at Hypoxia Condition in vitro

作　　者：杨雪[1] 王志荣[1] 张卓琦[1] 张超群[1] 徐晤[1] 程明月[1] 

机构地区：[1]徐州医学院附属医院心内科,江苏省徐州市221002

出　　处：《中国循环杂志》2013年第8期622-626,共5页Chinese Circulation Journal

基　　金：江苏省卫生厅医学重点人才基金(RC2007089)

摘　　要：目的:探讨白藜三醇(Res)对氯化钴(CoCl2)诱导缺氧的人脐静脉内皮细胞(HUVEC)的血管生成作用的影响及可能机制。方法:体外培养HUVEC分5组:DMEM高糖(<4500 mg/L)培养基培养细胞组(正常组)、CoCl2+DMEM高糖培养基(缺氧组)、缺氧+LY294002(阻断剂组)、缺氧+白藜三醇(药物组)、缺氧+白藜三醇+LY294002(药物阻断剂组)。采用CCK-8法检测细胞增殖情况;采用蛋白免疫印迹(Western blot)法检测低氧诱导因子1α(HIF-1α)、血管内皮生长因子(VEGF)、蛋白激酶B(Akt)、磷酸化蛋白激酶B(p-Akt)的表达;免疫细胞化学的方法检测HIF-1α的蛋白表达;采用transwell chamber(迁移小室)检测内皮细胞的迁移能力;体外血管形成实验检测内皮细胞的体外血管形成能力。结果:①CCK-8:缺氧组及药物组(0.1、1、5、10μmol/L)与正常组相比A490值升高(P均<0.01);药物组(1、5μmol/L)与缺氧组比A490值升高(P均<0.01)。尤其药物组5μmol/L A490值明显升高(P<0.01)。②蛋白免疫印迹:缺氧组较正常组HIF-1α、VEGF、p-Akt蛋白表达均增加(P均<0.05);药物组(5μmol/L)与缺氧组相比HIF-1α、VEGF、p-Akt蛋白表达均增加(P均<0.01);药物阻断剂组(5μmol/L)较阻断剂组HIF-1α、VEGF、p-Akt蛋白表达比较差异均无统计学意义(P均>0.05)。③免疫细胞化学:缺氧组胞内HIF-1α阳性表达,显色呈黄褐色;药物组(5μmol/L)胞内HIF-1α强阳性表达,显色呈深黄褐色。④transwell chamber:药物组(5μmol/L)内皮细胞迁移率大于缺氧组(P<0.01)。⑤体外血管形成实验:药物组(5μmol/L)在体外的血管形成数量明显大于缺氧组(P<0.01)。结论:白藜三醇可能是激活磷脂酰肌醇3-激酶(phosphatidylinositol 3-kinase,PI-3K)/Akt通路促进HIF-1α聚集,从而促进血管的新生。Objective： To study the effect of resveratrol （Res） for improving the angiogenesis in human umbilical vein endothelial cell （HUVEC） at Coc12 induced hypoxia condition in vitro. Methods： The HUVEC were cultured in 5 groups, ①ontrol group, the cells were cultured with high glucose （〈4500 rag/L） DMEM, ② Hypoxia group, cells cultured with COC12, ③ Blocker group, cells cultured with COC12＋LY294002, ④ Medication group, cells cultured with CoC12＋Res at different concentrations, ⑤ Hypoxia＋medication＋blocker group. HUVEC proliferation was detected by CCK8 assay, the protein levels of hypoxia-inducibie factor-1 α （HIF-1α）, vessel endothelial growth factor （VEGF）, protein kinase B （Akt） and p-Akt were examined by Western blot analysis, HIF-1 α expression was measured by immunocytochemistry, cell migration rate was studied by transwell chamber method, and cell angiogenesis was examined by tube formation test. Results： ① By CCK8 assay, for HUVEC proliferation, Hypoxia group and Medication （0.1, 1, 5, 10 μmol/L） group had increased A490 level than that in Control group, and A490 level in Medication （1, 5 μmol/L） group was higher than that in Hypoxia group, all P〈0.O1. @） By Western blot analysis, HIF-1α, VEGF, p-Akt protein expression significantly increased in Hypoxia group than that in Control group, all P〈0.01. HIF-1α, VEGF and p-Akt protein expression increased in Medication （5 μmol/L） group than that in Hypoxia group, all P〈0.05. HIF-1α, VEGF, p-Akt protein expression were similar in Blocker group, all P〉0.05. ① By immunohistochemistry, HIF-1 α was positive in Hypoxia group, and strongly positive in Medication （5 μmol/L） group.④ By transwell chamber method, cell migration rate in Medication （5 μmol/L） group was higher than that in Hypoxia group, P〈0.01. ⑤ By tube formation test, the angiogenesis in Medication （5 μmol/L） group was more than that in Hypoxia group, P〈0.01. Conclusion： Res may activate Akt pathway,

关 键 词：白藜三醇 低氧诱导因子 血管内皮生长因子 蛋白激酶B 血管生成 

分 类 号：R541[医药卫生—心血管疾病]