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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 signaling in the inhibition of mitochondrial ATP-sensitive potassium channel (mito-KATP) opening in fractalkine (FKN) expression induced by hypoxia-reoxygenation in rat cardiac microvascular endothelial cells (MMECs). Methods The isolated MMECs from SD rats were cultured in vitro and were randomly divided into 4 groups (n = 6): normal control group (N group), hypoxia / reoxygenation group (H / R group) , Diazoxide pretreatment + hypoxia / reoxygenation group (DZ group), LY294002 + diazoxide preconditioning + hypoxia / reoxygenation group (LY294002 + DZ group). DZ group was pretreated with 100μmol / L diazoxide for 2h, LY294002 + DZ group pretreated with 100μmol / L LY294002 for 2h and 100μmol / L diazoxide preconditioned for 2h, then hypoxia 2h, reoxygenation 2h. The apoptotic cells were observed by Hoechst staining. The viability of apoptotic cells was measured by MTT assay. The levels of Akt and FKN mRNA were detected by RT-PCR and Akt protein by Western blotting. Results Compared with N group, the proliferation rate of H / R group was significantly decreased (P <0.01), the apoptosis rate was significantly increased (P <0.01), the expression of FKN mRNA and FKN protein was significantly increased (P <0.01) Akt protein increased (P <0.05). Compared with H / R group, the proliferation rate of DZ group was significantly increased (P <0.01), the apoptosis rate was significantly decreased (P <0.01), Akt mRNA and protein were significantly increased (P <0.01 and P <0.05) And protein expression decreased significantly (P <0.01). Compared with DZ group, Akt mRNA and protein in LY294002 + DZ group were significantly decreased (P <0.01), and FKN mRNA and protein expression were significantly increased (P <0.01). Conclusion Mito-KATP opening regulates the transcription and expression of downstream FKN mRNA through PI3K / Akt signaling, which promotes cell proliferation and inhibits cell apoptosis and protects against hypoxia-reoxygenation-induced MMECs injury.