论文部分内容阅读
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。
OBJECTIVE: To construct and identify the adenovirus expression vector carrying the N-terminal 1 ~ 76 fragment (CGA1-76) of human chromogranin A, vasostatin-1 (VS-1) gene and observe the effect of recombinant adenovirus in rat cardiomyocytes To investigate the protective effects of VS-1 transgenic mice on cardiomyocytes hypoxia / reoxygenation injury. Methods: (1) Using CGA1-58 cDNA template, the primers were designed and the cDNA of CGA1-76 was amplified by PCR. The cDNA of CGA1-76 was cloned into the shuttle plasmid pAdTrack of adenovirus. The recombinant plasmid was linearized with Pme I and ligated with adenovirus backbone plasmid pAdEasy- 1 homologous recombination in Escherichia coli BJ5183. After identification of the correct homologous recombination plasmid pAd-VS-1, QBI-293A cells were linearized and packaged. The recombinant adenovirus Ad-VS-1 was amplified and infected with protein H9c2 Western blotting was used to detect the expression. (2) Hypoxia / reoxygenation (H / R) injury model of cardiomyocytes was established and divided into 4 groups: blank group, H / R group, Dye + H / R group. The activity of cardiomyocytes, the activity of superoxide dismutase (SOD) and the content of malondialdehyde (MDA) in each group were measured. Results: (1) The recombinant plasmid pAd-VS-1 was confirmed by PCR, gene sequencing and restriction enzyme digestion. The recombinant plasmid pAd-VS-1 was successfully constructed and transfected into QBI-293A cells. -1 protein expression. (2) Compared with blank group, the content of MDA and the activity of SOD in H / R group were significantly lower than those in blank group. The VS-1 gene transfection increased the activity of myocardial cells and SOD in H / R model and decreased the production of MDA . CONCLUSION: Ad-VS-1 is successfully constructed and can infect rat cardiomyocytes to express VS-1 and protect H / R injury of cardiomyocytes. The mechanism is related to the anti-oxidative effect.