抑制RANKL诱导破骨细胞分化的DNA适配子的筛选

Identification of a DNA Aptamer Inhibiting RANKL-induced Osteoclastogenesis in Vitro

目的:筛选能高特异性、高亲和力结合RANKL蛋白并有效抑制RANKL对破骨细胞诱导分化作用的DNA适配子。方法:首先,采用原核系统表达并纯化RANKL蛋白,通过SELEX(Systematic evolution of ligands by exponential)技术从人工合成的单链随机寡核苷酸文库中筛选能高特异性、高亲和力结合RANKL蛋白的DNA适配子。然后,用RNAfolding sever software分析适配子空间结构,以ELISA检测DNA适配子和RANKL亲和力大小并筛选出亲和力最高的一组DNA适配子用以验证DNA适配子对RANKL诱导破骨细胞分化的抑制作用。结果:(1)成功在原核系统表达并纯化RANKL蛋白;(2)筛选出能高特异性、高亲和力结合RANKL蛋白的12个DNA适配子。(3)与对照组相比,不同浓度DNA适配子能明显抑制TRAP阳性破骨细胞数量(P<0.05),且浓度越高抑制效果越明显。结论:成功筛选出的DNA适配子能特异性结合RANKL蛋白并有效抑制RANKL对破骨细胞的诱导分化功能。

Objective： To select ssDNA aptamers with high affinity and specificity for RANKL which could efficiently inhibit RANKL-induced osteoclastogenesis in vitro. Methods： Firstly, a 93nt single stranded DNA （ssDNA） random library was subjected to 14 rounds of selection against RANKL expressed and purified from the prokaryotic expression system by SELEX method. And then the structure of ssDNA aptamers was analyzed by RNAfolding server soRware and the affinities of aptamers to RANKL were determined by ELISA. Lastly, the inhibition effects of ssDNA aptamers on RANKL-induce osteoclastogenesis were verified in vitro. Results： @Mouse RANKL was successfully expressed and purified by the prokaryotic expression system. @Twelve ssDNA aptamers with high affinity and specificity for RANKL were selected out. @the numbers of the TRAP＋ multinueleated osteoelasts were decreased in different groups of ssDNA aptamers comparing with control group（P〈0.05）. Conclusions： The ssDNA aptamers of RANKL have been successively selected by the SELEX method for the first time and the selected ssDNA aptamers can effectively inhibit RANKL-induced osteoclastogenesis in vitro.