识别多种种属来源间充质干细胞核酸适配体的筛选及鉴定

Selection and identification of aptamers for mesenchymal stem cells from different species

目的筛选能识别多种属来源的间充质干细胞(MSC)的核酸适配体,并对其进行鉴定。方法从幼兔股骨骨片分离MSC并进行成骨、成脂诱导分化鉴定。以分离得到的兔源性MSC为筛选靶标,利用细胞SELEX技术筛选核酸适配体。将第5轮富集文库进行克隆、测序,利用RNA structure、MEME软件分析获得候选序列,利用流式细胞仪鉴定候选适配体与兔、大鼠、人源性MSC的结合。结果从兔股骨骨片分离得到的MSC可诱导向成骨、成脂分化。经过5轮SELEX筛选获得兔MSC富集文库。候选适配体5-1-12不仅结合兔MSC,也识别大鼠和人源性MSC。结论活细胞SELEX筛选获得与多种属来源MSC结合的核酸适配体5-1-12。

Objective To screen an ss DNA aptamer for rabbit mesenchymal stem cells（ MSCs） and to identify the ability of the aptamer to recognize MSCs of a variety of species origin. Methods MSCs were isolated from the thigh bone of immature rabbits and identified by induced osteogenic and adipogenic differentiation,respectively. Aptamers were screened by cell SELEX（ systematic evolution of ligands by exponential enrichment） technique targeting to isolated MSCs.Enrichment of the 5^（th） pool was evaluated through binding assay of FAM modified pool to MSCs by confocal microscopy. The enriched 5^（th）pool was then cloned into p GE-T vector and the cloned sequences were determined randomly. The candidates were chosen based on primary sequence conservation and predicted secondary structure by RNA structure and MEME online analysis. Flow cytometry analysis was used to identify the aptamers binding to MSCs of rabbit,rat,and human origin.Results The isolated MSCs had the potential of osteogenic differentiation and adipogenic differentiation under certain conditions. Aptamer 5-1-12 from 5^（th）enriched pool was characterized as MSCs recognizing aptamer binding to MSCs of rabbit,rat and human origin. Conclusion Aptamer 5-1-12 that recognizes MSCs of different species origin is obtained through live cell-SELEX.