重组nkx2.5腺病毒抑制过氧化氢诱导的H9c2细胞凋亡

Recombinant adenovirus overexpressing nkx2.5 protects H9c2 cells against H2O2-induced apoptosis

摘要:

为研究心脏发育关键基因nkx2.5的功能及应用价值，构建Ad-Nkx2.5重组腺病毒，并检测nkx2.5过表达拮抗氧化应激损伤的效应及机制。采用AdEasy 腺病毒表达系统构建Ad-Nkx2.5重组腺病毒，建立H2O2诱导H9c2心肌细胞凋亡模型，分别用Ad-Nkx2.5重组病毒或对照病毒感染细胞，采用Hoechst33342染色观察细胞形态变化、MTT法检测细胞存活率，免疫印迹检测caspase-3活化、细胞色素C的胞浆含量。并通过Real-time PCR检测凋亡相关基因bcl-2和bax表达。结果发现，nkx2.5过表达促进H9c2细胞存活，抑制H2O2诱导的caspase-3活化及线粒体细胞色素C的释放。Nkx2.5过表达上调bcl-2表达，显著下调H2O2诱导的bax表达。并发现H2O2对Nkx2.5核定位无明显影响。结果显示重组腺病毒介导的Nkx2.5过表达可通过调控凋亡相关基因表达，抑制线粒体凋亡途径，保护心肌细胞抗氧化损伤。

Abstract:

To study the function and potential application of nkx2.5, a critical gene for heart development, we constructed a recombinant adenovirus overexpressing nkx2.5 gene (Ad-Nkx2.5) with the AdEasy system. To evaluate the effect and mechanism of Ad-Nkx2.5 against oxidative injury, the H9c2 myocardial cells were infected with the recombinant adenoviruses Ad-Nkx2.5 or Ad-EGFP, and subsequently exposed to H2O2 to induce apoptosis. The anti-apoptotic potential of Ad-Nkx2.5 was validated by MTT assay for cell viability, Hoechst33342 staining for cellular morphology, and immunoblotting for caspase-3 activity. Ad-Nkx2.5 infection led to an increased survival rate of H9c2 cells and decreased the amount of caspase-3 in an active form. Additionally, overexpression of Nkx2.5 inhibited the release of cytochrome C from the mitochondria into the cytosol. Mechanismic studies showed that Nkx2.5 upregulated bcl-2 gene expression and significantly repressed H2O2-induced expression of bax detected by Real-time PCR. Additionally, H2O2 treatment did not affect the nuclear localization of Nkx2.5. These findings indicate that adenovirus-mediated nkx2.5 gene transfer exerted a protective effect on H9c2 cells against H2O2-induced apoptosis via mitochondrial pathway, and the Nkx2.5-mediated expression modulation of apoptosis-associated genes could be involved in this event.