促红细胞生成素衍生肽抑制大鼠心肌微血管内皮细胞缺血再灌注损伤的作用研究

发布时间：2018-05-19 14:06

本文选题：促红细胞生成素衍生肽 + 心肌微血管内皮细胞　；参考：《中国循环杂志》2015年03期

【摘要】：目的:探讨促红细胞生成素衍生肽(HBSP)抑制大鼠心肌微血管内皮细胞(CMECs)缺血/再灌注损伤的作用及其机制。方法:分离培养大鼠CMECs,建立缺血/再灌注损伤模型,随机分为对照组、模拟缺血/再灌注组(SI/R组)、SI/R+重组人促红细胞生成素组(SI/R+rh EPO组)、SI/R+HBSP组;HBSP分别选择2.5 ng/ml、25 ng/ml、50 ng/ml、100 ng/ml四个浓度,通过噻唑蓝(MTT)比色实验检测CMECs增殖能力以确定药物作用的最适浓度。而后采用细胞划痕实验检测细胞迁移能力,末端脱氧核苷酸转移酶介导的d UTP缺口末端标记测定(TUNEL)法检测细胞凋亡率来进一步验证其保护作用。研究保护机制的实验分组为:对照组、SI/R组、SI/R+HBSP(HBSP最适浓度为25 ng/ml)组、SI/R+HBSP+磷脂酰肌醇-3激酶(PI3K)抑制剂(LY294002)组、SI/R+HBSP+雷帕霉素组,采用蛋白免疫印迹(Western blot)法检测蛋白激酶B(AKT)、哺乳动物雷帕霉素靶蛋白(m TOR)、核糖体S6蛋白激酶(p70S6K)等蛋白的表达及其磷酸化水平。结果:与SI/R组比,SI/R+rh EPO组和SI/R+HBSP组CMECs增殖能力明显上升(P0.05),凋亡率显著下降(P0.01),迁移能力增强。用抑制剂LY294002及雷帕霉素处理后,与SI/R组相比,SI/R+HBSP组AKT、m TOR及p70S6K的磷酸化水平均显著提高(P0.05);与SI/R+HBSP组相比,SI/R+HBSP+LY294002组的AKT、m TOR及p70S6K的磷酸化水平均显著下降(P0.05),SI/R+HBSP+雷帕霉素组m TOR磷酸化水平及p70S6K磷酸化水平显著下降(P0.05)。结论:HBSP能够抑制缺血/再灌注诱导的CMECs的损伤,其保护作用可能与PI3K-AKT/mT OR信号通路激活有关。
[Abstract]:Aim: to investigate the inhibitory effect of erythropoietin derived peptide (HBSP) on myocardial microvascular endothelial cells (CMECs) ischemia / reperfusion injury in rats and its mechanism. Methods: CMECs were isolated and cultured in rats. The model of ischemia / reperfusion injury was established in rats. The rats were randomly divided into four groups: the control group, the simulated ischemia / reperfusion group and the SI-R / R recombinant human erythropoietin group. The rats in the SIP-R / R HBSP group were divided into four concentrations of 2.5 ng / ml 25 ng / ml 50 ng / ml 100 ng/ml, respectively. The optimal concentration of CMECs was determined by MTT colorimetric assay. Then the cell migration ability was detected by cell scratch assay and the apoptosis rate was detected by terminal deoxynucleotidyl transferase-mediated d UTP Nick end labeling method to further verify its protective effect. The experimental groups to study the protective mechanism were as follows: the optimal concentration of SIR / R HBSP(HBSP in the control group was 25 ng / ml), and the SIR HBSP phosphatidylinositol 3 kinase PI3K inhibitor, LY294002, was used in the SIP-R HBSP rapamycin group. The expression and phosphorylation level of protein kinase (BK), mammalian rapamycin target protein (mTORA) and ribosomal S6 protein kinase (p70S6K) were detected by Western blot assay. Results: compared with SI/R group, the proliferative ability of CMECs in SIR rh EPO group and SI/R HBSP group was significantly increased (P 0.05), the apoptosis rate was decreased significantly (P 0.01), and the migration ability was enhanced. After treated with inhibitor LY294002 and rapamycin, Compared with SI/R group, the phosphorylation levels of p70S6K and p70S6K in Sirs / Sirs HBSP group were significantly higher than those in SI/R HBSP group, and the phosphorylation levels of TOR and p70S6K in Sirs / R HBSP LY294002 group were significantly lower than those in SI/R HBSP group, and the phosphorylation levels of m TOR and p70S6K in Sirs / Sirs HBSP rapamycin group were significantly lower than those in SI/R HBSP group. Conclusion CMECs injury induced by ischemia / reperfusion can be inhibited by 1: HBSP, and its protective effect may be related to the activation of PI3K-AKT/mT OR signaling pathway.
【作者单位】：中国人民解放军第四军医大学第一附属医院心内科;
【基金】：国家自然科学基金(81200100)
【分类号】：R965

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