摘要
目的:探究白藜三醇(Res)对正常状态下人脐静脉内皮细胞(HUVEC)血管生成的影响及其可能机制。 方法:体外培养HUVEC,实验分为:DMEM高糖(〈4500 mg/L)培养基培养组(正常组)、白藜三醇组(药物组)、白藜三醇+磷脂酰肌醇3-激酶(PI3K)阻断剂组(LY294002,药物阻断剂组1)、LY294002组(阻断剂组1)、白藜三醇+NG-硝基-L-精氨酸甲酯盐酸盐组(一氧化氮合酶阻断剂L-NAME,药物阻断剂组2)、L-NAME组(阻断剂组2),其中LY294002为磷脂酰肌醇3-激酶阻断剂。采用CCK-8法检测细胞增殖情况;免疫印迹(Western blot)法检测蛋白激酶B(Akt)、磷酸化蛋白激酶B(p-Akt)、内皮型一氧化氮合酶(eNOS)、磷酸化内皮型一氧化氮合酶(p-eNOS)蛋白的表达;亚硝酸还原酶法检测一氧化氮(NO)的水平;迁移小室(transwell chamber)检测内皮细胞的迁移能力;小管形成实验检测内皮细胞的体外小管形成能力。 结果:①药物组(1、5μmol/L)与正常组相比细胞增殖增加(P〈0.01);药物组(5μmol/L)与药物组(0.2、10、20μmol/L)相比细胞增殖增加(P〈0.01);药物组(20μmol/L)对细胞增殖有抑制作用(P〈0.01),差异均有统计学意义。②药物组(5μmol/L )较正常组p-Akt、p-eNOS蛋白表达增加(P〈0.05~0.01),NO水平增加(P〈0.05),差异均有统计学意义;药物阻断剂组1 p-Akt、p-eNOS蛋白表达与药物组比较均减低(P均〈0.01),NO水平减低(P〈0.05);药物阻断剂组1(5μmol/L)较阻断剂组1 p-Akt蛋白、p-eNOS蛋白及NO差异均无统计学意义(P均〉0.05)。③细胞迁移及小管形成实验:药物组(5μmol/L)内皮细胞迁移率及体外小管形成数量大于正常组(P〈0.01),药物阻断剂组2内皮细胞迁移率及体外小管形成数量小于药物组(P〈0.01)。 结论:白藜三醇可能通过激活PI3K/Akt/eNOS信号通路诱导正常状态下内皮细胞NO水平的增加,从而促进内皮细胞增殖、迁移及体外小管形成。
Objective: To explore the effect of resveratrol (Res) on angiogenesis of human umbilical vein endothelial cells (HUVEC) with the possible mechanisms in vitro. Methods: The HUVECs were cultured in 6 groups.①Control group, HUVEC were cultured with high glucose in DMEM,②Res group, the cell were cultured with Res at different concentrations,③Res with PI3K blocker LY294002 (Res+Blocker 1) group, ④Blocker 1 group, HUVEC were cultured with LY294002 alone, ⑤Res with eNOS blocker L-NAME (Res +Blocker 2) group and ⑥Blocker 2 group. The effect of Res on HUVEC proliferation was detected by CCK-8 kit, the protein expressions of p-Akt, p-eNOS were examined by Westin blot analysis, nitric oxide (NO) level was measured by nitrate reduction method and the endothelial cell migration was assayed by transwell chamber method. Results: ① Compared with Control group, HUVEC proliferation increased in Res (1, 5μmol/L ) group, P〈0.01, the proliferation in Res (5μmol/L) group was higher than those in Res (0.2, 10, 20μmol/L) group, P〈0.01, while Res (20 μmol/L) group could inhibit the proliferation P〈0.01. ②Compared with Control group, Res (5μmol/L) group had the higher protein expressions of p-Akt, p-eNOS, P〈0.05-0.01, higher NO level, P〈0.05.③Compared with Res group, Res+Blocker 1 group had lower expressions of p-Akt, p-eNOS, P〈0.01, lower NO, P〈0.05; the expressions of p-Akt, p-eNOS and NO level were similar between Res+Blocker 1 group and Blocker 1 group, all P〉0.05.④Compared with Control group, the cell migration and tubing formation were higher in Res (5μmol/L) group, P〈0.01;compared with Res group, the cell migration and tubing formation were lower in Res+Block2 group, P〈0.01. Conclusion: Res could up-regulate NO level via activating PI3-K/Akt/eNOS signaling and therefore, improving the proliferation, migration and tubing formation of HUVEC in vitro.
出处
《中国循环杂志》
CSCD
北大核心
2014年第8期643-647,共5页
Chinese Circulation Journal