Establishment Of The Evaluation Method Of Exosome Isolation And Purification

Exosomes are one of the membrane vesicles secreted to extracellular matrix by many cells.It's plays important role in cell communication and system adjustment through carrying lipids and protein on membrane and loading cargos(neurotransmitterproteins.hormones and cytokines etc.)in cell.According to different secretory pathway,extracellular vesicles(EVs)including exosomes(30-100 nm)and microvesicles(100-1,000 nn).Researchers found exosomes exist widely in cell culture media,body fluids such as blood,milk,urine and saliva,the kind and number is closely bound up with the body's physical states.Exosome is expected to become the new types of biomarker for disease diagnosis,drug nanocarriers,treatment reagents and drug targets,it has broad application prospect in the field of diseases diagnosis and therapeutics.Due to exosome exist in the complex biological environment and exosome derived from different cells have significant differences in size,morphology and biochemical characteristics.As a result,effective separation and detection methods of exosome are still two major problems of exosome research.The current common methods of exosome purification are differential ultracentrifugation,size exclusion,exosome precipitation and immunoaffinity.Characterized these nano-vesicle at single particle level is of great importance to study on mechanism of vesicle transport systems and cell communication,early diagnosis and treatment of diseases.At present,the single particle detection methods of exosome is cryo-TEM.nanoparticle tracking analysis(NTA).tunable resistive pulse sensing(TRPS),but the latter two difficult to detect the exosome whose size is less than 70 nm and can only obtain size distribution and concentration of particles.Flow cytometry is advanced detection technique for rapid,multi-parameter and quantitative analysis of individual cells or cell-size particles in suspension.However,because of the limitation of detection sensitivity traditional flow cytometry is difficult to achieve analysis nanoparticles smaller than 200 nm and fluorescence sensitivity smaller than 500 FITC.Exosome is mainly distributed in 30-120 nm,the amount of protein and nucleic acid expression are relatively low,so the traditional flow cytometry can't realize the detection requirement for exosome.Our laboratory successfully developed High Sensitivity Flow Cytometry(HSFCM),detection of single silica nanoparticles with 24 nm in diameter.And the fluorescence detection limit is single phycoerythrin fluorescent molecules.HSFCM offering a rapid detection rate which up to 10 000 particles per minute,and can realize the scattering and multiple fluorescence of single particle at the same time.Therefore,HSFCM satisfy the detection needs for exosome.Based on the excellent detection sensitivity of HSFCM,this dissertation optimized the condition of ultracentrifuge extract exosome.Comparison of ultracentrifuge and commercial kits in exosome concentration,size distribution,purity and recovery.This paper aimed to give researchers an efficient and standardized method for exosome isolation and purification,reducing the ambiguity findings resulting from the use of different purification methods.This dissertation includes the following parts:In chapter one,the research background of exosome and the important application in disease diagnosis,therapeutics of exosome,exosome isolation methods and detection techniques re briefly introduced.Chapter two introduces the particle size and concentration analysis of exosome isolated from plasma by different methods.Ultracentrifugation is the most common method to isolate exosome,but the condition of centrifuge is still unknown.We choose the optimum condition for ultracentrifuge purification exosome through optimization.The scattering light intensity of exosome from different isolation methods were detected based on the scattering light sensitivity of HSFCM.Using polystyrene fluorescent microsphere with known concentration as external standard,we can obtain the concentration of exosome at the same condition of HSFCM.Make comparison of concentration of exosome from plasma and vesicle-depleted plasma.According to the law of Rayleigh scattering,the scattering light intensity of particle is not only related to the particle size but also related to the particle material.Using silica nanoparticles as the size reference standards needs refractive correction.Then according to size and scattering intensity of silica nanoparticle,we can draw a standard working curve of scattering intensity and particle size.Then we can obtain the size distribution of exosome.It found out that exosome from plasma mainly distributed in 40-100 nm,and plasma with high particle concentration whereas concentration of exosome isolated by ultracentrifugation is very low.Chapter three demonstrates the purity?morphology and recovery of exosome from different isolation methods.Exosome shows great application prospect in cancer diagnosis,prognosis and treatment.Because of lacking efficient isolation and detection method,restrict the clinical application of exosome.According to exosome purification,quality control is significant,the existence of impurities can influence downstream analysis.The purity of exosomes can be measured by using Triton X-100 to lyse the membrane of vesicles and analyze the particle concentrations before and after the detergent treatment via single particle enumeration.At the same time,we use exosome from cell culture medium as exosome standard to measure the recovery of exosome isolation methods.HSFCM is an advance technology for simple and rapid detection of exosome,and HSFCM requires less sample volume,short detection time,so it's provide an efficient detection method for exosome development.It turned out that exosome isolated by ultracentrifugation have the most purity exosome and the typical morphology characterized by TEM.Chapter four describes the protein analysis of exosome from different methods.Exosome have a lot of proteins and receptor,CD9,CD63 and CD81 are currently recognized as exosome biomarkers.We use immunofluorescence staining analysis the protein expression of exosome from plasma.Then use SDS-PAGE characterize the total protein of exosome from plasma and vesicle-depleted plasma.At last,exosome biomarkers CD9,CD63 and CD81 were analyzed by western blot.In chapter five,the work of this thesis is summarized and the future prospect in exosome analysis is discussed.