首页|利用生物素-链亲和素系统筛选呼吸机所致肺损伤HMGB1启动子结合蛋白

利用生物素-链亲和素系统筛选呼吸机所致肺损伤HMGB1启动子结合蛋白

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目的:筛选呼吸机所致肺损伤(VILI)高迁移率族蛋白B1(HMGB1)的启动子结合蛋白.方法:制备VILI大鼠模型,与正常对照组大鼠同期处死,取肺组织提取细胞核蛋白.用PCR法扩增末端带生物素标记的HMGB1启动子探针,将细胞核蛋白与制备的HMGB1启动子探针进行孵育,再以标记有链亲和素的磁珠分离HMGB1启动子-蛋白反应复合物,分别用0.25 moL/L和1 moL/L NaCl洗脱探针上结合的蛋白,SDS-PAGE电泳分离样品蛋白,并对凝胶进行银染显色,比较VILI组与正常对照组的差异条带.结果:两组细胞核蛋白中共观察到24条差异条带.分析差异条带显示,与正常对照组比较,在低亲和力作用水平,机械通气后有6个蛋白与HMGB1启动子的结合力增强,有2个蛋白的结合力减弱;在高亲和力作用水平,有10个蛋白与HMGB1启动子的结合力增强,有6个蛋白的结合力减弱.结论:筛选到一些VILI特异性HMGB1启动子结合蛋白,对于研究HMGB1的转录调控机制具有重要意义.
Screening of binding proteins that interact with HMGB1 promoter in ventilator-induced lung injury by biotin-streptavidin system
AIM:To screen the binding proteins that interact with HMGB1 promoter in ventilator-induced lung injury(VILI).METHODS:Eight Sprague Dawley(SD)rats were divided into two groups,the mechanically ventilated group(n=4)and the normal control group(n=4).The mechanically ventilated rats were subjected to mechanical ventilation(VT=40 mL/kg)for 4 hours to reproduce a ventilator-induced lung injury(VILI)model.The biotin-labeled DNA probe for HMGB1 promoter was amplified by PCR.The cell nuclear extracts from lung of rats were prepared and mixed together.DNA pull-down assays were performed by using magnetic beads conjugated with streptavidin.Proteins binding on the beads were eluted with different concentrations of salts.The samples were resolved by SDS-PAGE and displayed by silver stain to compare differential bands between two groups.RESULTS:Twenty four proteins in the nuclear extracts were found to be difierent between normal and VILI rats.SDS-PAGE displayed that,compared with normal rats,there were six proteins inereased and two declined in the rats treated with mechanical ventilation when eluted with lOW salt.While under the condition of high affinity elution,there were ten proteins increased and six disappeared after mechanical ventilation.CONCLUSION:Binding proteins that interact with HMGB1 promoter in VILI have been acquired.which will be useful for further study of the regulation mechanism of HMGB1.

high mobility group box 1 proteinmechanical ventilationacute lung injurypromoterbiotin-streptavidin

丁宁、肖慧、许立新、余守章

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广州市第一人民医院麻醉科和麻醉学实验室,广东,广州,510180

广州市第一人民医院门诊部,广东,广州,510180

高迁移率族蛋白B1 机械通气 急性肺损伤 启动子 生物素-链亲和素

广东省医学科学研究基金广州市医药卫生科技一般引导项目广州市医药卫生科技一般引导项目广州市医药卫生科技重点项目

A20094972008-YB-0122009-YB-0212008-ZDi-14

2009

第四军医大学学报
第四军医大学

第四军医大学学报

CSTPCDCSCD北大核心
影响因子：0.599
ISSN：1000-2790
年,卷(期)：2009.30(21)
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