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8型腺相关病毒介导短发夹状RNA在小鼠肝脏的表达及其功能 被引量：1

Study of adeno-associtated virus serotype 8 mediated shRNA delivery and function in livers of mice
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摘要 目的以内源性基因超氧化物歧化酶1(SOD1)为靶基因,观察经单次尾静脉注射后8型腺相关病毒(AAV8)介导的短发夹状RNA(shRNA)在小鼠肝脏内的表达、对靶基因的沉默效应以及重组AAV8-SOD1 shRNA的副反应。方法利用AAV8载体系统的双启动子构建能同时表达绿色荧光蛋白(GFP)和SOD1 shRNA的重组AAV8-SOD1 shRNA。采用Northern blot和Western blot分别检测SOD1shRNA在小鼠肝脏内表达水平、靶基因SOD1在mRNA和蛋白水平表达变化、肝细胞微小RNA122(miRNA-122)表达水平;标准酶法测定小鼠肝功能;酶联免疫实验测定干扰素-α(IFN-α)水平;经HE染色对肝组织进行病理学观察。结果经尾静脉注射重组AAV8-SOD1 shRNA后,小鼠肝组织中可检测到SOD1 shRNA和SOD小干扰RNA(SODsiRNA)反义链的表达;靶基因SOD1在mRNA和蛋白水平表达均明显受抑;血清转氨酶轻度升高,但IFN-α水平无上调;肝组织无明显病理学改变;肝细胞miRNA-122水平无明显下降。结论经尾静脉单次注射重组AAV8-SOD1 shRNA后,SOD1 shRNA后可在小鼠肝脏中高效表达并沉默靶基因表达。重组AAV8-SOD1 shRNA对小鼠机体无明显副反应。 Objective To investigate the expression of short hairpin RNA （shRNA） mediated by adeno-associated virus serotype 8 （AAVS） in the livers after a single injection from tail vein of ICR mice. At the same time, the suppression of endogenous target gene, superoxide dismutase （SOD1） and the side effect of recombinant AAV8-SOD1 shRNA were observed. Methods The AAV8 vector was designed to express both green fluorescent protein （GFP） driven from the CMV promoter and a shRNA targeting SOD1 driven from the U6 promoter. The expression of SOD1 shRNA, SOD1 mRNA and miRNA122 in liver were detected by Northern blot, while the expression of SOD1 protein in livers were evaluated by Western blot. Standard enzyme methods were employed in detecting the liver function and enzyme linked immunosorbent assay （ELISA） was used to detect the serum level of interferon α （ IFN α）. At the same time, liver tissue sections were stained with HE method and the pathological changes were observed. Results Northern blot proved that SOD1 shRNA could be expressed and processed to antisense strand of siRNA in livers after a single injection from tail vein. Western blot and Northern blot analyses evinced that the expression of SOD1 mRNA and protein were significantly suppressed in liver. Liver function test showed that there was a mild increase of transaminases at 2 weeks. But serum IFN α level was normal. Histological examination didn＇ t show any abnormal changes in liver. And the levels of miRNA-122 were not decreased in mice expressing shRNAs. Conclusion AAV8 can efficiently deliver shRNA into liver and suppress the expression of endogenous target gene. Recombinant AAV8 has little side effect, therefore it could be a useful gene delivery vector.
作者 陈颖伟 久保寺 李一 大西威一郎 仁科一隆 李定国 横田隆德
机构地区 上海交通大学医学院新华医院消化内科 日本东京医科齿科大学神经内科
出处 《上海交通大学学报（医学版）》 CAS CSCD 北大核心 2007年第12期1438-1443,共6页 Journal of Shanghai Jiao tong University:Medical Science
关键词 AAV8 SHRNA RNA干扰 超氧化物歧化酶1 adeno-associated serotype 8 shRNA RNA interference SOD1
分类号 Q78 [生物学—分子生物学]
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参考文献12

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二级参考文献29

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上海交通大学学报（医学版）
2007年 第12期

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