【摘要】 目的：探讨细胞穿透肽PEP-1介导的人过氧化氢酶(CAT)对H9c2细胞缺氧复氧损伤的保护作用及其可能的机制。方法：利用基因工程手段表达和纯化His-tag-PEP-1-CAT及His-tag-CAT融合蛋白。将H9c2细胞随机分为正常对照组(CTL组)、缺氧复氧组(H/R组)、H/R加CAT组(CAT组)、H/R加PEP-1-CAT组(PEP-1-CAT组)。分别向PEP-1-CAT组及CAT组加入2 mol/L的PEP-1-CAT及CAT蛋白500μL处理6 h,将各组细胞置于缺氧箱中缺氧21 h,再复氧6 h,通过CCK-8法监测细胞的增殖率，流式细胞仪检测细胞的凋亡水平和活性氧(ROS)的水平，DCFH-DA探针检测细胞内的ROS变化水平，JC-1试剂盒检测细胞线粒体膜电位的变化。Western blot分析凋亡蛋白Bcl-2、Bax、Caspase-3及p-p38表达量的变化。结果：与H/R组相比，PEP-1-CAT组细胞凋亡率、细胞内ROS水平、Bax、Caspase-3及p-p38表达量均明显下降(P<0.05),细胞增殖率、细胞内线粒体膜电位水平及Bcl-2的表达量升高(P<0.05)。结论：PEP-1-CAT对H9c2细胞缺氧复氧损伤有保护作用，推测机制可能与其抗氧化、抗凋亡作用有关，PEP-1-CAT可通过抑制p38MAPK信号通路阻断Bcl-2/Bax/线粒体凋亡通路。更多还原

【Abstract】 Objective To investigate the protective effect of the PEP-1-CAT fusion protein on hypoxia/reoxygenation-induced H9c2 cells and its possible mechanisms. Methods His-tag-PEP-1-CAT and His-tag-CAT fusion proteins were expressed and purified by genetic engineering. H9c2 cells were randomly divided into four groups: control(CTL) group, hypoxia/reoxygenation(H/R) group, H/R plus CAT treatment(H/R+CAT) group, and H/R plus PEP-1-CAT treatment(H/R+PEP-1-CAT) group. The PEP-1-CAT and CAT proteins(500 μL) were added at a concentration of 2 mol/L for 6 h, and the cells were then placed in a hypoxia chamber for 21 h, followed by reoxygenation for 6 h. The proliferation of H9c2 cells was monitored by the CCK-8 assay. Apoptosis and reactive oxygen species(ROS) were analyzed using flow cytometry. The changes in intracellular ROS were evaluated using DCFH-DA probes, and mitochondrial membrane potential changes were measured by the JC-1 assay kit. Western blot was performed to detect the expressions of apoptosis-related proteins Bcl-2, Bax, Caspase-3, and p-p38. Result Compared with the H/R group, the PEP-1-CAT group had decreased apoptosis, intracellular ROS, Bax, Caspase-3, and p-p38(P<0.05), while the proliferation rate, mitochondrial membrane potential, and Bcl-2 were elevated(P<0.05). Conclusion The PEP-1-CAT fusion protein protects hypoxia/reoxygenation-induced H9c2 cells, and the mechanism may be related to its antioxidant and anti-apoptotic properties through the inhibition of the p38MAPK signaling pathway, thereby blocking the Bcl-2/Bax mitochondrial apoptosis pathway.更多还原