Selection Of Aptamers For QBC-939Based On Cell-SEKEX

Hilar cholangiocarcinoma (HCCA) is a common and malignant tumor in bileduct system. The surgical therapy of HCCA is of great difficulty because the tumorgerminating site is special, and it has invasive growth and closely related with hilarvessels, which cause failure for tumor resection. At present the clinical findings anddiagnosis of hilar cholangiocarcinoma is mainly based on morphology imagingexamination of tissue and cells, which lacks high sensitivity and specificity.Therefore, the development of molecular probe with high specificity and highsensitivity for hilar bile duct cancer is very important for cancer early detection anddiagnosis, tumor marker discovery, and cancer pathogenesis studies. Aptamers are anew type of molecular probe developed in recent years. It demonstrates adavantagesin many characteristics such as a wide range of targets, good stability, smallmolecular weight, easy modification, and so on. Aptamers are now widely applied inanalytical chemistry and biomedical fields.In this paper, we adopted cell-SELEX for rapid screening of aptamers that canspecifically recognize human bile duct carcinoma cell line with humancholangiocarcinoma cell line QBC-939as target cells and human hepatoma cellSMMC-7721as control cells. The main contents are as follows:(1) selection of aptamers for human cholangiocarcinoma cell line QBC-939based on cell-SELEX strategyBased on cell-SELEX strategy, human cholangiocarcinoma cell line QBC-939was used as target cell line, and human hepatocellular car cinoma cell lineSMMC-7721cells was used as control cell line. Selection conditions were optimized,and the stringency was gradually increased during selection process by increasing theproportion of target and library, reducing the incubation time, incre asing the washingstrength, and increasing the negative selection strength. After14repeated screening,we verified the target recognition ability of selected pools, and the result showed thatthe sequences that can recognize QBC-939were highly enriched in the pool, and the13th selected pool were cloned and sequenced.(2) aptamer optimization and aptamers binding sites investigationOn the basis of the work in first chapter, we used Clustal X2software tostimulate the secondary structure of the selected aptamer sequences, and classifiedthem into6different families based on sequence similarities in secondary structure.Six representive sequence from different families were subjected to binding assay, and among them two sequences demonstrated excellent binding affinity toward tatgetcells, specifically down to nanomolar level. Then preliminary researches wereconducted to investigate temperature adaptiveness of the two aptamers and theirbinding sites on cells. Finally, based on the difference and simil arities of thesecondary structures of the two aptamers, we abtained a optimized23nt aptamer withhigh affinity and specificity.