The Aptamers Selection Of Human Doxorubicin-resistant Hepatocellular Cancer And The Performance Research

The most important treatment for middle-late stage Hepatocellular carcinoma(HCC)is chemotherapy.However,the development of drug-resistance in HCC can dramatically reduce the efficacy of chemotherapeutic treatment.Early detection of drug-resistant hepatocellular carcinoma,discovery and research of drug-resistant hepatocellular carcinoma related molecules and the mechanisms regulating the development of drug resistance in HCC and reverse the drug-resistant could effectively reduce the resistance,and effectively extend the life of patients.It is able to abtain cell specific nucleic acid aptamers without previously knowing its target molecule by cell-SELEX technology.It provides a new strategy and idea for drug-resistant protein discovery and cancer clinical diagnosis and typing,and development mechanisms of drug resistance in HCC.In this work,aptamers were selected with drug-resistant hepatocellular carcinoma as the positive cell,and the hepatocellular carcinoma cell line as the negative cell based on Cell-SELEX.Abtained aptamers were characterized and put into preliminary application.1.Aptamer selectionBased on the Cell-SELEX,drug-resistant hepatocellular carcinoma cell line HepG2/ADM as target cell and hepatocellular carcinoma cell line HepG2 as control cells.In order to achieve the aptamer with high specificity and high affinity,the stringency of selection condition was gradually increased.Finally,only 12 rounds of selection were performed,the enrichment of the selection library reach the plateau.After cloning and sequencing,four sequences were obtained.And further characterization shows that sequence H1 of four sequences could specifically recognize target cell.2,Sequence analysis and primary application of the aptamers for human drug-resistant hepatocellular carcinomaSubsequently,we optimized the sequence H1 structure based on the analysis of secondary structure by the software Mfold and then a truncated aptamer d3 with 50 nts were achieved with equilibrium dissociation constant as low as 7 nM.Characterization shows that truncated sequence d3 have the advantages of high specificity,high affinity,and high temperature stability by flow cytometry.In addition,according to the results of the treatment of protease,we speculated that the target molecule on the cell surface is likely to be a membrane protein,which provide a strong foundation to study resistant protein and drug resistance of drug-resistant hepatocellular carcinoma cells,and is expected to be applied to the early diagnosis of drug-resistant cell and molecular targeted therapy.3.Research of resistant cells resistance and reversing drug resistance of liver cancerFinally,the target molecule of aptamer d3 and the drug resistance of drug-resistant hepatocellular carcinoma cells was investigated,including binding sites,targets and the drug resistance.The experimental results show that the target molecule of aptamer d3 is not resistant protein Pgp,but related to the drug resistance of resistant cells and may be another resistant protein on the cell.In addition,we also investigated the influence of aptamer on drug resistance.Research findings show that the aptamer combined with drug-resistant hepatocellular carcinoma cells can reduce the drug resistance of cells,advantageous to the treatment of drug-resistant cells.