| 摘要: |
| 目的:构建并鉴定携带人嗜铬粒蛋白A的N端1~76片段(CGA1-76) 即vasostatin-1（VS-1）基因的腺病毒表达载体,观察重组腺病毒在大鼠心肌细胞中的表达,探讨VS-1转基因治疗对心肌细胞缺氧/复氧损伤的保护作用。方法:（1）利用CGA1-58cDNA模板,设计引物,利用PCR合成、扩增CGA1-76的cDNA并将其克隆到腺病毒穿梭质粒pAdTrack,经PmeⅠ酶切线性化后与腺病毒骨架质粒pAdEasy-1在大肠埃希菌BJ5183中同源重组。将鉴定正确的同源重组质粒pAd-VS-1线性化后转染QBI-293A细胞进行包装、扩增得到重组腺病毒颗粒Ad-VS-1,将该病毒感染大鼠心肌细胞H9c2并通过蛋白质印迹法检测其表达。（2）建立心肌细胞缺氧/复氧（H/R）损伤模型,分为4组:空白组、H/R组、空载腺病毒转染+H/R组、Ad-VS-1转染+H/R组；测定各组心肌细胞活力,超氧化物歧化酶（SOD）活性和丙二醛（MDA）含量。结果:（1）经PCR、基因测序、酶切鉴定证实重组质粒pAd-VS-1构建成功,将其导入QBI-293A细胞,通过蛋白质印迹法证实病毒颗粒Ad-VS-1感染心肌细胞后VS-1蛋白表达。（2）H/R组心肌细胞活力和SOD含量较空白组明显降低,MDA增加；而VS-1基因转染提高了心肌细胞H/R模型心肌细胞活力和SOD含量，并降低了MDA生成量。结论:成功构建Ad-VS-1,将其感染大鼠心肌细胞后能够表达VS-1并对心肌细胞H/R损伤具有保护作用,其机制和抗氧化作用有关。 |
| 关键词: Vasostatin-1 基因重组；腺病毒；缺氧/复氧；心肌保护 |
| DOI：10.3724/SP.J.1008.2009.0491 |
| 投稿时间：2008-09-01修订日期：2009-02-04 |
| 基金项目: |
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| Protective effect of adenovirus-mediated vasostatin-1 gene transfection on rat cardiomyocyte hypoxia/reoxygenation injury |
| WANG Zheng,XIAO Ming-di*,YU Min,YUAN Zhong-xiang |
| (Department of Cardiothoracic Surgery,Shanghai First People’s Hospital affiliated to Shanghai Jiaotong University,Shanghai 200080,China) |
| Abstract: |
| Objective:To construct and identify adenovirus vector containing the chromogranin A 1-76 segment (CGA1-76),or vasostatin-1 (VS-1) gene,and to observe its expression in cardiomyocytes of rats, so as to investigate the protective effects of VS-1 transgenic therapy on myocardial hypoxia/reoxygenation (H/R) injury. Methods:(1) The primers of CGA1-76 cDNA was designed and used for PCR amplification. The products were then cloned into adenovirus shuttle plasmid pAdTrack and linearized by enzyme Pme Ⅰ; the resultant plasmid was co-transfected into E.coli BJ5183 cells with adenovirus backbone plasmid pAdEasy-1 for homologous recombination. Then the recombinant plasmid was identified,linearized and packaged with QBI-293A cells to amplify the recombinant adenovirus Ad-VS-1,which was then used to infect H9c2 cardiomyocytes. Western blotting was used to detect the expression of VS-1 protein expression. (2)Myocardial H/R model was established and the cells were divided into 4 groups:blank group,H/R group,mock infection + H/R group,and Ad-VS-1 infection + H/R group. The viability of cardiomyocytes,the activity of superoxide dismutase (SOD) and the content of malondialdehyde (MDA) were measured. Results:(1) The recombinant plasmid was constructed successfully as confirmed by PCR,sequencing and enzyme digestion. Western blotting confirmed the protein expression of VS-1 in the H9c2 cells. (2)The viability of cardiomyocytes and the activity of SOD in H/R group were obviously lower than those of the blank group; the level of MDA was higher than that of the blank group. Transfection with VS-1 increased the cardiomyocytes viability and SOD activity in the H/R model group and decreased the production of MDA. Conclusion:Ad-VS-1 has been successfully constructed,and transfection with it can protect rat cardiomyocytes from H/R injury,which is related to the anti-oxidation process. |
| Key words: vasostatin-1 gene recombination adenovirus hypoxia/reoxygenation myocardial protection |
