Detection Glioblastoma Multiforme Cells With Aptamer Cocktails And Selection Of DNA Aptamers Against Glioma Exosomes

Objective:1.To enhance the recognition sensitivity of glioblastoma cell line T98 G using the aptamers cocktail.2.Selecting a high-affinity aptamer as a novel molecular probe for identification and sorting of glioma exosomes.Methods:1.The synergistic effect of aptamers WYZ-41 a and WYZ-50 a obtained by the Cell-SELEX technique were identified by flow cytometry.The result of confocal images demonstrates colocalization of WYZ-41 a and WYZ-50 a on T98 G.Meanwhile,detecting the stability and binding ability of aptamers in the physiological environment by FACS to further investigate its potential to practical application.2.Exosomes secreted by glioma cells SHG44 purification procedure based on differential ultracentrifugation.The biological and morphological characteristics were verified by flow cytometry and transmission electron microscopy.Using the aptamer library of glioma cell line SHG44,aptamer with the strongest binding ability to SHG44 exosomes was obtained by flow cytometry analysis.Simulate its secondary structure by software,truncate and optimize it by retaining the main stem and loop structure.The exosomes produced by other glioma cell lines were purified,and the aptamer selectivity was analyzed by flow cytometry.Using flow cytometry test binding capacity of aptamer in the physiological environment.Using the characteristics of the aptamer to bind the target,its bound target was detected by mass spectrometry.Result:1.There is a synergistic effect between aptamers WYZ-41 a and WYZ-50 a to bind to the T98 G cell membrane.The result of laser confocal microscopy indicated aptamers WYZ-41 a and WYZ-50 a partially overlap on the cell membrane.It has good stability in serum and cerebrospinal fluid with maintaining great binding capacity and specificity for target cells in serum and cerebrospinal fluid.2.The exosomes of glioma cell lines SHG44 were isolated and purified by differential ultracentrifugation.The result of electron microscopy showed exosomes with a bilayer membrane structure and diameter in the range of 40-100 nm.Flow cytometry showed positive expression of the exosome-specific protein marker CD63.Flow cytometric analysis of glioma cell lines SHG44 aptamer library was performed,in which the aptamer S12 had the best affinity and specificity.By predicting the secondary structure of the aptamer,the truncation was optimized with preservation of the major stem and loop structure.The truncated aptamer S12-4 can maintain good binding ability and specificity for target cells in physiological environments such as serum and cerebrospinal fluid.Incubation with pre-treated trypsin exosomes,S12-4 lost its original binding ability.Aptamer S12-4 modified biotin was used to preliminarily identify biomarker of SHG44 exosomes.Conclusion:1.Aptamers WYZ-41 a and WYZ-50 a have a synergistic effect and are colocalized on the cell membrane.They have good stability under physiological conditions and good binding ability to target cells and can be used as a molecule of glioblastoma.Probes will play an important role in early diagnosis,targeted drug delivery,and improved prognosis of glioblastoma.2.The obtained aptamer S12-4 has high specificity and affinity,and can distinguish glioma SHG44 exosomes from other exosomes of glioma cell lines and can be used as a sorting probe for exosomes.The preliminarily identified biomarker is heat shock protein 70(HSC70),which is expected to play a unique role in the early diagnosis of glioma and the evaluation of tumor prognosis.