SELEX技术筛选钙通道特异性核酸适配体及其抗肥大细胞活化的作用  

Calcium channel-specific oligonucleotide aptamers selected by SELEX inhibit mast cell activation

作　　者：杨永强[1] 陈晓红[2] 李怡然[2] 陈波[2] 冉新泽[3] 杨涛[1] 孙仁山[1] 

机构地区：[1]第三军医大学大坪医院野战外科研究所皮肤科,重庆400042 [2]第三军医大学药学系药理学教研室,重庆400038 [3]第三军医大学军事预防医学院全军复合伤研究所创伤烧伤与复合伤国家重点实验室,重庆400038

出　　处：《第三军医大学学报》2014年第4期307-312,共6页Journal of Third Military Medical University

基　　金：国家自然科学基金(30972647)~~

摘　　要：目的筛选针对Orai1分子第1膜外区蛋白的核酸适配体,探讨适配体通过拮抗钙离子释放激活通道(Ca2+release activation channel,CRAC)抑制肥大细胞活化的效应。方法构建随机单链DNA文库,利用不对称PCR对单链DNA文库进行扩增。采用聚苯乙烯酶联板为介质的指数富集配体系统进化(systematic evolution of ligand by exponential enrichment,SELEX)技术筛选核酸适配体。利用酶联免疫吸附实验检测各轮筛选序列与靶蛋白的亲和力,验证适配体与靶蛋白的特异性结合;激光共聚焦显微镜观察适配体对细胞内钙离子浓度变化的影响;利用IgE介导的肥大细胞脱颗粒模型,观察适配体对β-氨基己糖苷酶释放的抑制效应。结果经过12轮SELEX筛选,获得了7条核酸适配体,其中AptamerY1解离常数(Kd值)为1.72×10-8mol/L。以终浓度2μg/mL的AptamerY1可抑制由IgE介导的人肥大细胞系LAD2钙离子内流,并对LAD2细胞的β-氨基己糖苷酶释放抑制率达95%。结论通过SELEX筛选,获得了7条针对Orai1分子第1膜外区的核酸适配体,所获得适配体可对肥大细胞LAD2钙内流有效抑制,进而抑制肥大细胞活化和β-氨基己糖苷酶的释放。Objective To screen the oligonucleotide aptamers for the first extracellular domain of Orai1 protein in order to determine the inhibitory effects of aptamers on mast cells activation by Ca2＋ release activation channel （CRAC）.Methods Random single-strand DNA （ssDNA） library was constructed. The key parameters of asymmetric PCR were optimized to amplify ssDNA library. The oligonucleotide aptamers were screened by systematic evolution of ligand by exponential enrichment （SELEX） technology with enzyme-linked polystyrene plate as medium. The affinity between ssDNA library and target protein was examined in each round by ELISA. The changes of intracellular Ca2＋ concentration in LAD2 mast cell line were tested by laser confocal microscopy. The β-hexosaminidase release from LAD2 cells was measured so that the aptamer effects on mast cell activation mediated by IgE could be evaluated.Results Seven oligonucleotide aptamers were obtained after 12 rounds of SELEX. The Kd value of Aptamer Y1 reached 1.72×10-8 mol/L. Aptamer Y1 at the final concentration 2 μg/mL decreased the calcium entry and inhibited the β-hexosaminidase release from LAD2 cells by 95%.Conclusion The seven oligonucleotide aptamers targeting the first extracellular domain of Orai1 protein are obtained by SELEX technology. The aptamers can decrease intracellular calcium influx and inhibit the release of β-hexosaminidase from LAD2 cells effectively, and therefore, inhibit mast cell activation through CRAC channels.

关 键 词：肥大细胞 SELEX适体技术 钙离子释放激活通道 β-氨基己糖苷酶 

分 类 号：R392.33[医药卫生—免疫学] R392.12[医药卫生—基础医学]