ABSTRACT
Background and purpose:As the development of the completion of the human genome project (HGP), the research focus is turning to the gene function research. At present, the domestic experimental research on the apoptosis of K562 cells induced by antisense olignonucleotides is rare. This study was aimed to investigate the effect of human chronic myelogenou leukemia (CML) bcr-abl fusion gene antisense oligonucletides on autophagy and apoptosis of CMLK562 cells in vitro. Methods:By liposome as the carrier, K562 cells were transfected with the bcr-abl gene antisense olignonucleotides. Hoechst staining method was used to observe the apoptosis inducing effect of different concentrations of oligonucleotides, the expressions of LC3-Ⅱ, autophagy-related protein, were determined by the Western blot method, the cell cycles were determined by lfow cytometry (FCM), and JEM-4000EX electron microscope technology was used to detect the apoptosis morphological changes. The apoptosis was detected by DNA agarose gel electrophoresis. Results:Hoechst staining results showed that the bcr-abl gene antisense oligonucletides signiifcantly promoted the apoptosis of K562 cells in a certain concentration dependent manner. Western blot showed that the expression level of LC3-Ⅱwas obviously higher in bcr-abl gene antisense oligonucletides transfected group than the control group, showing a promoting effect on cell autophagy. FCM test results showed that bcr-abl gene antisense oligonucleotides transfected K562 cells showed obvious cell cycle arrest, visible obvious apoptosis morphology under the electron microscope, and DNA Ladder showed obvious apoptosis fragments. Conclusion:The bcr-abl gene antisense olignonucleotides can signiifcantly induce the cell apoptosis of K562. This study provides a new method for CML therapy.
ABSTRACT
Objective:To screen and characterize aptamers against BCR-ABL fusion protein.Methods:A 90bp single stranded DNA( ssDNA) random library was subjected to 13 rounds of selection against BCR-ABL fusion protein by systematic evolution of ligands by expotential enrichment ( SELEX ) method, the selected aptamers were cloned and sequenced.The primary sequences and structure of aptamers were analyzed by Clustal W and DNA Folding Sever and the percentage of the ssDNA pool bound to BCR-ABL core protein were determinated.Results: after 13 rounds selection, the percentage of ssDNA pool bound to BCR-ABL fusion protein increased from 0.3%to 47.1%,the results showed that affinities of the Aptamers were different,the second structure analysis revealed possible stem-loops for binding to BCR-ABL fusion protein,the affinity of aptamer A2 to BCR-ABL fusion protein was highest with Kd values as low as 72 nmol/L.Conclusion:Aptamers against BCR-ABL fusion protein has been identified by SELEX methods from a 90 bp single stranded DNA library.And provide certain reference for the clinical treatment of chronic myelogenous.