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

doi:10.3969/j.issn.2095-4344.2016.40.014

Abstract

Abstract:

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 mRNA 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 mRNA 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.

中国组织工程研究杂志出版内容重点：肾移植；肝移植；移植；心脏移植；组织移植；皮肤移植；皮瓣移植；血管移植；器官移植；组织工程

Key words: Genes, Cells, Diabetic Cardiomyopathies, Tissue Engineering

CLC Number:

R318

Li Su-juan, Yuan Wen-chang, Mai Yun-pei, Hou Ning. Adenoviral vectors carrying Brahma-related gene 1 attenuates oxidative stress-induced apoptosis of cardiomyocytes[J]. Chinese Journal of Tissue Engineering Research, 2016, 20(40): 6021-6027.

Figures/Tables 1

2.1 重组腺病毒载体Adeno-Brg1转染心肌细胞的效率原代培养新生大鼠心肌细胞转染Adeno-Brg1 24 h后，可见Adeno-Brg1转染组和空载体Adeno-EGFP转染组被转染的细胞搏动规律有力，透光度好，见图1。荧光显微镜下观察可见，Adeno-Brg1转染组细胞核内绿色荧光明显增强，提示核内Brg1表达增高，阳性细胞转染率达90%以上，而空载体Adeno-EGFP转染组绿色荧光仅见于胞浆中，见图2。说明构建的重组腺病毒能感染心肌细胞，对细胞活性无明显影响。 2.2 重组腺病毒载体Adeno-Brg1对心肌细胞Brg1 mRNA表达的影响分别利用RT-PCR、Real-time RT-PCR检测转染后Brg1 mRNA表达。转染24 h后，与对照组相比，Adeno-Brg1 mRNA表达量增加7.8倍(P < 0.01)；空载体Adeno-EGFP对心肌细胞Brg1 mRNA表达无明显影响(P > 0.05)，见图3。 2.3 重组腺病毒载体Adeno-Brg1对心肌细胞Brg1蛋白表达的影响 Western-blot检测可见Brg1、GAPDH特异性条带，相对分子质量分别为210 000、37 000左右。转染Adeno-Brg1后心肌细胞目的蛋白表达显著高于空载体Ad-EGFP转染后心肌细胞的Brg1表达，见图4。 2.4 H2O2对心肌细胞Brg1表达的影响 100 µmol/L 的H2O2处理12 h，Western-blot结果显示，H2O2可显著抑制心肌细胞Brg1表达，见图5。 2.5 重组腺病毒载体Adeno-Brg1对H2O2诱导的心肌细胞凋亡的影响腺病毒转染24 h后继续给予 100 µmol/L H2O2处理12 h，流式细胞仪检测细胞凋亡率。结果显示，H2O2处理组能明显诱导心肌凋亡，细胞凋亡增加175%；而Adeno-Brg1转染组，细胞凋亡率降低35%；单纯腺病毒转染组和空载体Ad-EGFP转染组对细胞凋亡无明显影响，见图6。 Western-blot结果显示，H2O2显著诱导凋亡标志蛋白cleaved-Caspase3表达，Adeno-Brg1转染后能显著抑制H2O2诱导的cleaved-Caspase3表达，结果与流式细胞仪检测结果一致；单纯腺病毒转染组和空载体组对cleaved-Caspase3表达无影响，见图5。"

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