Research On Fluorescence Analysis And Imaging Methods Of Exosome Membranes

Exosomes are cell-secreted membrane-coated vesicles,and their sizes are variable from 30 to 150 nm.Exosomes contain a variety of proteins,DNA,coding and non-coding RNA,lipids and other substances derived from host cells,which are closely related to tumor formation,growth,metastasis and disease occurrence.Therefore,exosomes have become hot spot in research of tumor diagnosis and treatment.However,accurate analysis and detection technology for membrane structure of exosome are very limited,which restricts the scientific research and clinical research of exosomes.In this thesis,membranes of exosome were used as research object,and the research of fluorescence analysis and fluorescence imaging method were carried out.(1)Considering the wide sizes range of exosomes,in order to achieve accurate analysis,the exploration of segmentation separation technology was carried out.In this thesis,a variety of separation techniques were tried,including PEG precipitation,size exclusion chromatography and ultrafiltration.Finally,the scheme was determined as physical membrane filtration.On the basis of ultrafiltration,the segmented separation of exosomes derived from cell culture media with different sizes was achieved.The exosomes were labeled with green fluorescent protein(EGFP),and the EGFP-labeled exosomes were taken up by the recipient cells.The experiment verified that the exosomes separated by PEG precipitation and ultrafiltration maintained normal functional activity.This provides usable experimental materials for research on exosomes.(2)At present,common quantitative method of exosomes is based on the total protein content analysis of exosomes,which is cumbersome and easily interfered by various impurity proteins.In this paper,the lipid on exosome membranes was first proposed as the object of quantitative analysis,and the total membrane lipid assay(MLA)of exosomes was established.The specific strategy was to quantify exosomes by fluorescence analysis technology combined with two fluorescent dyes through ratio fluorescence analysis.This strategy required dye A to be a specific membrane dye that was sensitive to membrane of exosomes,and dye B was a water-soluble dye that was insensitive to membranes,which served as an internal reference dye for dye A.Therefore,a membrane-specific dye Mem-SQAC was selected as dye A from the four lipophilic membrane dyes of PKH26,Mem-BDP,Mem-CA and Mem-SQAC,which was characterized by high anti-protein interference ability,high anti-lipoprotein interference ability and high signal-to-noise ratio of fluorescence imaging.Rhodamine sulfonate was selected as dye B from two kinds of rhodamine dyes.It had been applied to the quantitative analysis of exosomes derived from MCF-7 cells,MCF-10A cells,HeLa cell culture medium and breast cancer patients' serum,and it was found that the amounts of exosomes contained in serum was more.MCF-7 cells were easier to secrete exosomes than HeLa cells and MCF-10A cells.It was proved that this quantitative method was fast,convenient and low-cost.This provides a usable method and tool for rapid and accurate quantification of exosomes.(3)The sizes of exosomes are closely related to their functions,but there is no feasible and accurate imaging method to determine the sizes of exosomes.Based on the relationship between the membrane viscosity and the curvature radius of exosomes,this paper proposed a new strategy to evaluate the sizes of exosomes by measuring the membrane viscosity of exosomes.High-resolution fluorescence lifetime imaging(FLIM)was combined with membrane-specific viscosity-sensitive fluorescent probe of Mem-BDP,and the sizes of exosomes were reflected by measuring the fluorescence lifetime value(local viscosity)on exosome membranes.Firstly,the exosome samples with different sizes(10-50,50-100 and 100-220 nm)derived from HeLa cells and MCF-7 cells were stained and labeled with Mem-BDP,and fluorescence lifetime imaging(FLIM)was used to establish the relationship between the sizes of exosomes and the fluorescence lifetime values.The results showed that the average fluorescence lifetime value(average viscosity)of exosomes decreased with the increase of exosome sizes.Secondly,this method was used to perform fluorescence lifetime imaging(FLIM)on two groups of tumor cells and corresponding normal cells and serum-derived exosomes from different types of breast cancer patients.The results showed that the average fluorescence lifetime(average viscosity)of exosomes from tumor cells was higher than that of corresponding normal cells,and the average fluorescence lifetime value(average viscosity)of exosomes derived from the serum of different breast cancer patients were different.This provides a feasible method for precise measurement of the sizes of exosomes.(4)The labeling method is a key factor,which affect the accuracy of super-resolution imaging for exosomes.This paper proposed a new strategy to optimize the accuracy of superresolution imaging for exosomes.The specific strategy was using protein fusion technology to label exosomes with fluorescent proteins.Firstly,recombinant plasmids of fluorescent protein fused with marker protein were constructed to verify the feasibility of fluorescent protein labeling exosomes.Secondly,the application of three different exosome labeling methods(membrane dye,immunofluorescence,fluorescent protein)in receptor cell uptake imaging were compared,and the results showed that fluorescent protein labeling was the optimal method.Fluorescent protein labeling had the characteristics of accurate imaging results and unaffected original biological activity of exosomes.Finally,by labeling exosomes with light-activated fluorescent protein(PAmcherry),super-resolution imaging of extracellular and intracellular exosomes was realized.This provides a feasible tool for non-destructive,real-time,and visual imaging of exosomes.