Screening Techniques For Human Bone Microvascular Endothelial Cell Aptamers

In 2003,when severe acute respiratory syndrome swept the world and had a huge impact,patients were treated clinically with a large number of hormones such as corticosteroids,but after following up on those who recovered from SARS,it was found that some patients developed ischaemic necrosis of the femoral head to varying degrees during the recovery process,with a probability of 60～70%,and this hormone-induced osteonecrosis of femoral head is the main cause of non-traumatic osteonecrosis of femoral head necrosis.The pathogenesis of non-traumatic femoral head necrosis is still unclear,but the basic feature is impaired microcirculation of local blood to the bone.Therefore,bone microvascular endothelial cells,which constitute the microvascular lining in the bone and are involved in the formation of microcirculation,are rapidly becoming a hot topic of current research.In this study,we introduced capillary electrophoresis-based exponential enrichment ligand phylogeny(CE-SELEX)to the traditional cell-based exponential enrichment ligand phylogeny technique(Cell-SELEX),and successfully screened aptamers with high affinity for bone microvascular endothelial cells.The screening was performed with the help of the efficient separation ability of CE,and the sequences bound to the target cells were targeted and collected.7 candidate sequences were obtained after 4 rounds of screening through the key steps of library amplification by polymerase chain reaction(PCR)technique,purification of amplification products by asymmetric PCR method,and characterization by agarose gel electrophoresis.The screening process was monitored by quantitative real-time fluorescence PCR(q RT-PCR),and molecular mock docking of the candidate sequences was performed by MOE software to assist in the identification process.Finally,the sequence with high affinity(111.86 ± 18.36 n M)was selected as the aptamer(T-24)by comparing the affinity of each candidate sequence by flow cytometry analysis.This study focuses on human somatic cells that should be emphasized by SELEX research,and constructs a more complete CE-Cell-SELEX screening model for cell screening.This screening mode eliminates the need for negative screening,eliminating the traditional elution step and greatly reducing the screening cycle.The aptamer obtained from the screening can be used as a biological probe for the identification and isolation of bone microvascular endothelial cells due to its high specificity,high affinity,small molecular weight and non-immunogenicity,which is also beneficial to clarify the pathogenesis of non-traumatic femoral head necrosis,further construct and improve the research system of bone microvascular endothelial cells,and has certain reference significance for the early and rapid diagnosis and precise treatment of femoral head ischemia.