作者机构：
[唐标] Medical School, Hunan University of Chinese Medicine, Changsha 410028, China;[邓常清] Medical School, Hunan University of Chinese Medicine, Changsha 410028, China. dchangq@sohu.com
通讯机构：
[Deng, Chang-Qing] Medical School, Hunan University of Chinese Medicine, Changsha 410028, China.
作者机构：
[陈伶利; 宋岚; 张晓青; 钱纯; 李杰; 葛金文] Medical school of Hunan University of Chinese Medicine, Changsha 410208, China
关键词：
适体,核苷酸;tolC蛋白,大肠杆菌;抗药性
摘要：
目的:筛选大肠埃希菌外排泵tolC蛋白的核酸适配体。方法:重组表达大肠埃希菌外排泵外膜蛋白tolC,利用指数富集的配体系统进化技术(stematic evolution of ligand by exponential enrichment,SELEX)从单链DNA文库中筛选出一组能够特异性与其结合的核酸适配体。利用FITC荧光素标记技术检测每轮单链DNA文库与靶蛋白的亲和力,直至亲和力的上升趋于饱和,将最后一轮筛选产物克隆测序,并用DNAman软件分析其二级结构。结果:经过12轮筛选,单链DNA文库与靶标蛋白的亲和力趋向稳定,将第12轮筛选产物克隆测序,对获得的23个适配体进行分析。一级结构分析显示23个适配体并无共同的保守序列,但分别有3对和2对适配体序列完全一致。二级结构预测分析表明,茎环结构为适配体主要的结构形式,提示其可能是适配体与tolC蛋白蛋白特异性结合的基础。根据二级结构特点可将23个适配体分为4个家族,其中20号适配体与靶标蛋白亲和力最高。结论:成功利用SELEX技术筛选获得了特异结合tolC的高亲和力的核酸适配体,为大肠埃希菌耐药干预及机制研究奠定了基础。
摘要：
To explore the possible mechanism of Jiawei Danshen Yin（加味丹参饮）in treating cardiac reperfusion injury.Methods H9C2 cardiac muscle cells in vitro were randomized into 6 groups,with 6 multiple pores in each group.The blank serum group was cultured in culture medium containing 15％ of blank serum for 29 h.The hypoxia preconditioning group was cultured in hypoxia medium for 20 min,and then was given oxygen for 20 min,following another hypoxia culture for 20 min.After then the cells were moved into blank serum medium and routinely cultured for 24 h,following cultured in hypoxic medium for 2 h,and then given oxygen for 3 h.The hypoxia/reoxygenation group was cultured in culture medium containing 15％ of blank serum for 24h,then was moved into hypoxia medium and cultured in hypoxia condition for 2 h,then in oxygenation condition for 3 h.The serum containing medicine group was cultured in culture medium containing 15％ of medicine serum in normal condition for 24 h,and then was in hypoxia condition for 2 h,following in oxygenation condition for another 3 h.The serum containing medicine + autophagy inhibitor group was preconditioned by 10mmol/L 3-methyladenine for 30 min;the following treatment steps were the same as the serum containing medicine group.The serum containing medicine + autophagy agonist group was preconditioned by 100 nmol/L rapamycin for 30 min;the other treatment steps were the same as the serum containing medicine group.Growth and form changes of cardiac muscle cells in each group were examined with inverted microscope.The leakage rate of lactate dehydrogenase (LDH) in cell culture superuatant in each group was detected with colorimetric method.The form and number changes of myocardial cell autophagosome were examined with transmission electron microscope.The expressions of autophagy related protein LC3 Ⅱ and Beclin1 in each group were detected with Western blot method.Results Compared with that in the blank serum group,the degree of degeneration and necrosis of myocardial cells increased under microscope,there were a few autophagosome under electron microscope,the leakage rate of LDH in medium increased,and the level of Beclin1 protein increased in the hypoxia/ reoxygenation group (P ＜ 0.05 or P ＜ 0.01).Compared with the hypoxia/reoxygenation group,the degree of degeneration and necrosis of myocardial cells decreased,the number of autophagosome increased under electron microscope,the leakage rate of LDH in medium decreased obviously,and the expressions of LC3 Ⅱ and Beclin1 protein increased in the serum containing medicine group and the serum containing medicine + autophagy agonist group (P ＜0.05).Conclusion Jiawei Danshen Yin might strengthen autophagy by up-regulate the expressions of autophagy related protein LC3 Ⅱ and Beclin1 to protect injured cardiac muscle cells.
作者机构：
[宁毅] The Medicine School of Hunan University of Chinese Medicine, Changsha, 410208, China;[陈珂珂; 唐亮; 贺气志; 罗怀青; 徐倩] School of Basic Medical Science, Changsha Medical University, Changsha, 410219, China
