| Exosomes are lipid nanoparticles secreted by almost all cells and present in various body fluids.Through the specific formation mechanism,exosomes can carry the components of donor cells and be released into the extracellular,which is considered as an important way for cells to discharge garbage and communicate with the outside.Many studies have shown that exosomes can interact with nearby or distant cells based on the kind of their cargo,and allowing their cargo to function in recipient cells.The different functions of exosomes are mainly based on the donor cells by regulating the secreted exosome composition and quantity according to their own state.Therefore,as a natural nanovesicle released by cells,duo to their components can not only reflect the state of cells,but also transport cargoes between cells,exosomes have potential as a natural nano-drug delivery carrier and diagnosis and treatment markers of tumor.Although exosomes have great potential and advantages as tumor marker and drug delivery carrier,the studies about them are still immature.Therefore,exploring the role of exosomes in tumor diagnosis and treatment is still improtant.Based on the advantages of exosomes as the biomarke of tumor diagnosis and drug delivery carrier,the application of exosomes in the diagnosis and treatment for leukemia is studied.The work is divided into the following parts:First,the proteomic analysis of several leukemic cell lines derived exosomes found that nucleolins have the potential to be one of the markers of leukemia-associated exosomes.Secondly,based on the recognition of nucleolin on the exosomes,a dual-signal amplified fluorescence biosensor was designed to detect leukemia-related exosomes.Third,through the fusion expression of different membrane localization sequences with mCherry,the loading amounts of different fusion proteins in exosomes were analyzed,and then an optimal membrane localization sequence was selected to improve the sorting amount of mCherry in exosomes.Finally,based on the results of above membrane localization sequence analysis,a light-inducible system was designed to enrich intracellular RNA into exosomes,and the feasibility of exosomes as RNA drug delivery system was verified by experiments in vitro.The experimental contents and results as follows:(1)Exosomes secreted by several leukemia cell lines were collected by density gradient centrifugation,and their protein were identified by mass spectrometry.The results showed that exosomes derived from different cell lines had different protein expression,but all of them expressed nucleolin.Combined with flow cytometry,it was further demonstrated that nucleolin existed on the surface of leukemia-related exosome,and not on the surface of normal cell-derived exosome.Therefore,nucleolin may be one of the marker of leukemia-related exosomes.(2)Combined the recognition of aptamer,magnetic enrichment,and rolling circle amplification(RCA)based on dual signal amplification technology,an ultrasensitive fluorescent biosensor for the detection of leukemia-related exosomes was developed.Firstly,the products of RCA based double signal amplification were analyzed by agarose gel electrophoresis,indicating this method is feasibility.Then,the fluorescence intensity with or without signal amplification in the detection system was compared,it was further proved that the system could enhance the exosome detection signal.Finally,the specificity and sensitivity of sensor for exosome detection were analyzed under the optimized sensing parameters.The results showed that exosomes as low as 1 x 10~2/L could be detected with good specificity.In addition,exosomes in serum sample also could be successfully analyzed by this method.(3)Four different membrane localization sequences(Palm,PB,CAAX,CD63)were fused with mCherry,and the amount of mCherry selected into exosomes were analyzed.First,the expression plasmids of mCherry fused with three different membrane localization peptides and one transmembrane protein were constructed,and the corresponding stable cell lines were established.By analyzing the fluorescence imaging of mCherry in cells,it was proved that different membrane localization sequences make mCherry to distribute on the different location of cells.After collecting exosomes secreted by the different stable cell lines,western blot and flow cytometry were used to analyze the expression level of mCherry in exosomes.The results showed that the loading amount of mCherry in the exosomes was significantly different,and the loading amount of mCherry fused with CD63 and Palm was relatively high.In addition,the reason for the differential sorting of mCherry in exosomes was further analyzed by SIM imaging.(4)A light-inducible system for controlling exosomes to package and deliver endogenous RNA was constructed.The loading of endogenous RNA in exosomes was mainly based on the molecular interactions and the designability and biogenesis of exosome.First,three expression plasmids of Palm-EGFP-CIBN,CRY2-mCherry-MCP and BFP-miR-21sponge-MS2 were constructed,and the corresponding stable cell lines were established.Confocal microscopy of stable cell lines demonstrated that the protein and RNA expressed by the plasmids could be localized on the membrane through the molecular interactions(MS2-MCP,CIBN-CRY2)and regulation of blue light.Secondly,the analysis of EGFP and mCherry in exosomes with flow cytometry and SIM imaging further proved that the regulation of blue light could enrich above proteins in exosomes.In addition,the quantitative analysis of miR-21 sponge by real-time fluorescence quantitative PCR showed that the loading of mi R-21 sponge in exosomes was increased by 14 times.Finally,after modifying exosomes with aptamer AS1411,experiments in vitro showed that efficience of exosomes targeted into leukemia cells was enhanced.Functional experiments also showed that exosomes transported functional miR-21 sponges into leukemia cells,blocking the function of mi R-21 in leukemia cells,leading to the significant apoptosis of leukemia cells. |
PDF Full Text Request