PI3K/Akt在线粒体ATP敏感性钾通道开放抑制缺氧复氧大鼠心肌微血管内皮细胞FKN表达中的作用  

Role of PI3K/Akt signal pathway in opening mito-KATP to inhibit the expression of FKN in rat myocardium microvascular endothelia cells exposed to hypoxia-reoxygenation

作　　者：陈秋萍[1] 黄赛赛[1] 周伟伟[1] 沈施仁[1] 曹苏[1] 

机构地区：[1]南通大学附属医院麻醉科,江苏南通226001

出　　处：《解剖学报》2012年第6期778-783,共6页Acta Anatomica Sinica

基　　金：南通大学自然科学基金资助项目(10Z048);南通市科技基金资助项目(HS2011052)

摘　　要：目的观察PI3K/Akt信号在线粒体ATP敏感性钾通道(mito-KATP)开放抑制缺氧复氧大鼠心肌微血管内皮细胞(MMECs)fractalkine(FKN)表达中的作用。方法培养SD大鼠离体MMECs,建立缺氧复氧损伤模型24皿,随机分为4组(n=6):正常对照组(N组)、缺氧/复氧组(H/R组)、二氮嗪预处理+缺氧/复氧组(DZ组)、LY294002+二氮嗪预处理+缺氧/复氧组(LY294002+DZ组)。DZ组加入100μmol/L二氮嗪预处理2h,LY294002+DZ组在加入100μmol/L LY294002预处理2h后再加入100μmol/L二氮嗪预处理2h,然后和缺氧复氧组同样进行缺氧2h、复氧2h。Hoechst染色观察凋亡细胞显微结构,四甲基偶氮唑盐(MTT)法测定细胞活力,RT-PCR检测Akt和FKNmRNA水平,Western blotting检测Akt蛋白水平。结果与N组比较,H/R组细胞增殖率显著降低(P<0.01)、凋亡率显著升高(P<0.01),FKN mRNA和FKN蛋白表达显著增加(P<0.01)、Akt mRNA和Akt蛋白升高(P<0.05)。与H/R组比较,DZ组细胞增殖率显著升高(P<0.01)、凋亡率显著降低(P<0.01),AktmRNA和蛋白显著升高(P<0.01和P<0.05),FKN mRNA和蛋白表达显著降低(P<0.01)。与DZ组比较,LY294002+DZ组Akt mRNA和蛋白显著降低(P<0.01)、FKN mRNA和蛋白表达显著升高(P<0.01)。结论mito-KATP开放通过PI3K/Akt信号调节下游FKN mRNA的转录及表达,促使细胞增殖,抑制细胞凋亡,实现对缺氧复氧MMECs损伤的保护。Objective To investigate the role of PI3K/Akt signal pathway in openned mito-KATP inhibit the expression of fractalkine （FKN） in rat myocardium microvascular endothelial cells exposed to hypoxia-reoxygenation. Methods Cardiac microvascular endothelial cells of the SD rat were cultured to establish a hypoxia-reoxygenation model. Twenty-four dishes were randomly divided into 4 groups （n = 6 each）. The normal control group （group N ） , the cells of H/R group, diazoxide pretreatment ＋ H/R group （group DZ） and diazoxide pretreatment ＋ LY294002 ＋ H/R group （group DZ ＋ LY294002）. The cell vitality, the cell apoptotic rate and expression of Akt, FKN mRNA were detected with MTT. Hoechst dyeing RT-PCR and Western blotting, respectively. Results Compared with group N, the cell vitality significantly decreased （P 〈 0.01 ） and the apoptosis rate increased（ P 〈 0.01 ）. The expression of FKN mRNA and protein （P 〈 0.01 ） was up-regulated in H/R group, the expression of Akt mRNA and protein significantly rised （P 〈 0.05 ）. Compared with group H/R, the cell vitality significantly increased （P 〈 O. 05 ）, the apoptosis rate significantly decreased （ P 〈 0. 01 ）. The expression of Akt mRNA and protein （ P 〈 0. 01 and P 〈 0.05 ） was up-regulated, fractalkine mRNA andsignificantly decreased （P 〈 0. 01 ） in group DZ. Compared with group DZ, Akt protein and mRNA significantly decreased （ P 〈 0. 01 ）, FKN mRNA and protein significantly increased （ P 〈 0. 01 ）. Conclusion Openned mito-KATP through the PI3K/Akt signal pathway may regulate downstream of the transcription and expression of fractalkine mRNA inhibit cell apoptosis and improve the proliferation rate to protect the rat cardiac microvascular endothelial cells exposed on hypoxia- reoxygenation.

关 键 词：线粒体ATP敏感性钾通道 心肌微血管内皮细胞 PI3K Akt FRACTALKINE 缺氧 复氧 反转录-聚合酶链反应 免疫印迹法 大鼠 

分 类 号：R318.5[医药卫生—生物医学工程]