过表达Brahma-相关基因1腺病毒载体对氧化应激诱导心肌细胞凋亡的影响

Adenoviral vectors carrying Brahma-related gene 1 attenuates oxidative stress-induced apoptosis of cardiomyocytes

背景:Brahma-相关基因1是调节染色质重构的重要核蛋白,在糖尿病心肌病中表达降低。目的:构建大鼠Brahma-相关基因1重组腺病毒载体,评价其在氧化应激诱导原代心肌细胞凋亡中的保护作用。方法:构建Brahma-相关基因1重组腺病毒载体质粒,经人胚肾293T细胞包装、扩增、计算滴度后,将Brahma-相关基因1重组腺病毒转染入SD乳鼠原代培养心肌细胞,以空载体EGFP转染细胞及正常细胞作为对照,24 h后,荧光显微镜观察转染效率;荧光定量PCR、免疫印迹法检测细胞Brahma-相关基因1mR NA、蛋白表达;24 h后,分别加入100μmol/L H2O2处理12 h,免疫印迹法检测Brahma-相关基因1蛋白表达、凋亡标志物cleaved-Caspase3表达,流式细胞仪检测细胞凋亡率。结果与结论:(1)构建的重组腺病毒成功转染至心肌细胞,转染效率达90%以上,被转染细胞的Brahma-相关基因1 mR NA、蛋白表达明显增高;(2)H2O2可显著抑制心肌细胞Brahma-相关基因1表达,诱导心肌细胞凋亡,刺激心肌细胞cleaved-Caspase3表达;Brahma-相关基因1重组腺病毒可减轻由H2O2导致的上述心肌细胞损伤;(3)结果表明,氧化应激能直接抑制心肌细胞Brahma-相关基因1表达,Brahma-相关基因1在氧化应激诱导的心肌细胞凋亡中发挥保护作用。

BACKGROUND： Brahma-related gene 1（Brg1）, a catalytic subunit of an important chromatin remodeling complex, has been considered as a key nuclear transcriptional factor, and tends to be decreased in diabetic cardiomyopathy. OBJECTIVE： To construct an adenovirus vector carrying Brg1, and observe its protective role in oxidative stress induced-cardiomyocyte apoptosis. METHODS： The recombinant adenovirus plasmid was linearized and transfected into HEK293 cells using Fugene HD for packaging and amplification. The adenovirus particles were further purified, quantified, and sequentially transfected to cardiomyocytes of neonatal Sprague-Dawley rats. The Adeno-EGFP transfected and non-transfected cardiomyocytes were used as control group. 24 hours later, the transfection efficiency was observed by fluorescent microscope, and expressions of Brg1 mR NA and protein were detected by quantified PCR and western blotting. After treatment with 100 μmol/L H2O2 for 12 hours, the expressions of Brg1 protein and cleaved-Caspase 3 were measured by western blotting, and cell apoptosis was analyzed by flow cytometry. RESULTS AND CONCLUSION：（1） The recombinant adenovirus vector of Brg1 had been successfully transfected into cardiomyocytes with higher expressions of Brg1 m RNA and protein, and the transfection efficiency reached more than 90%.（2） After H2O2 treatment, the Brg1 was significantly down-regulated in contrast to the up-regulation of cleaved-Caspase 3; the flow cytometry data showed that the apoptotic cells were increased. But in Adeno-Brg1 transfected cardiomyocytes, the H2O2 induced cell apoptosis was significantly decreased compared with non-transfected cells and empty vector transfected cells.（3） These results suggest that oxidative stress can directly inhibit the Brg1 expression, and overexpression of Brg1 can protect the cardiomyocytes from cell apoptosis induced by oxidative stress.