| Exosomes are vesicles secreted by cells with a particle size of 30 nm-150 nm and a densit y of 1.10-1.18 g/m L,and contain specific lipids,protein,m RNA,mi RNA and DNA.It plays an important role in cell-to-cell communication.However,because of its small particle size,low abundance,low densit y,and the vesicles produced by cell secretion include not onl y exosomes,but also microcapsules with slight l y larger particle size,it is very difficult to separate them.At present,the commonl y used separation methods include ultracentrifugation,chemical precipitation and immune separation.However,these methods still have some disadvantages,such as ultrac entrifugation with high cost and low efficiency,chemical precipitation will introduce protein impurities,and immune separation will destroy the membrane structure of exosome and affect the biological activity of exosome.Efficient separation of exosomes still faces great challenges.In view of the problems existing in the separation and purification of exosomes,a magnetic separation method based on biological orthogonal reaction was designed in this paper.Dibenzocyc looctyne was activated by 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide,and then amidated with carbox yl groups on the surface of magnetic beads to obtain functional magnetic beads.N,N-dimethylethanolamine and 1,The reaction product of 2-dibromoethane and sodium azide continue to react to obtain azido ethyl choline.Then azido ethyl choline is connected to the cell membrane through click reaction.After a series of processes such as endocytosis,the exocrine membrane surface is provided with azido group s.The magnetic beads are connected with exocrine through bioorthogonal reaction.The exocrine is separated from the s ystem by magnetic separation.Then the phospholipid bond is hydrol yzed by alkaline phosphatase(ALP)to separate the magnetic beads from exocrine,thus obtaining a purer exocrine.Then,the exosomes extracted by this method were evaluated and identified by TEM,NTA,Western Blot and other experiments.The results showed that the exosomes extracted by this method were successfully extracted,and compared with those extracted by ultracentrifugation,the size of the exosomes extracted was more concentrated at about 100 nm,and the concentration was 4×10~5/m L.The protein on the exosomes was not damaged,which proved that this method did achieve high efficiency separation.In addition,exosomes also play an extremel y important role in the development of tumors.Previous studies on tumors found that the exocrine secreted by some cells in tumor tissues will increase the drug resistance of tumor cells and promote metastasis,etc.Tumor stem cells in tumor tissues will also secrete exocrine to affect common tumor cells,resulting in the failure of tumor treatment.However,at present,the exosomes secreted by cancer stem cells after chemotherapy have n o known effect on common tumor cells.Therefore,in order to explore the influence of exosomes of tumor stem cells on tumor therapy,the exosomes of tumor stem cells stimulated by chemotherapy were extracted b y functional magnetic beads,and the exosomes w ere incubated with common tumor cells to explore whether exosomes have an impact on common tumor cells and what impact they have.Through the experiments of cell vitalit y,balling abilit y,migration,invasion,HE staining,and expression of dryness markers,the dryness characterization of common tumor cells induced by exosomes was explored.It was found that exosomes secreted by tumor stem cells after chemotherapy can domesticate the surrounding common tumor cells and make them dedifferentiate into stem cells.This provides a new idea for tumor treatment.If the exocrine secretion of stem cells can be inhibited during treatment,the treatment efficiency will be greatl y improved. |
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