| BackgroundExtracellular vesicles(EVs)is a kind of nanoscale particles which secreted from cells.It contains the important biological information such as protein and RNA come from the source of cells.A growing number of studies shown that EVs play an important role in a variety of physiological and pathological process including the communication between cells,removing waste proteins from cells and the regulation of the immune mechanism.In addition,EVs has the specific biomarker which is expected to become a new biomarker of the disease diagnosis and monitoring.With the deeply research on EVs,the require with biochemical information of EVs are increaseing.The different kinds of human body fluids such as urine,plasma,serum during pregnancy,bronchial lavage fluid,joint synovial fluid,saliva and breast milk can secrete EVs.In addition,present studies have reported that a variety of different types of cells can secrete EVs,such as B lymphocytes,dendritic cells,mast cells,T cells,platelets,nerve cells and intestinal epithelial cells,retinal pigment epithelial cells,retinal cells,tumor cells and sperm.In 2004,for the first report that urinary extracellular vesicles(uEVs)can be successfully separated and identified in the urine by Pisitkun.He forecasted that uEVs are derived from a variety of urinary tract epithelial cells.Because a variety of corresponding trademark proteins of epithelial cells were found in EVsincluding glomerular podocyte,proximal tubule,pulp loops medulla thick wall section 1,manifold and bladder markers.According to the differences of density,size and specific markers of EVs,which include a variety of different types.The classification method of currently recognized by most people are according to the size of the uEVs and the different way of secretion.EVs broadly divided into three different kinds:the first one is exosome,which with the most widely research at present,the diameter of the exosome is 40?100 nm.The second one is microvesicles,for this kind of vesicles with the Intense controversy of the diameter range of the vesicle size,but most of the research that its diameter is from 100?100 nm.Apoptotic body is more than 1000 nm in diameter.The current research for uEVs is still in its infancy.It still needs to solve lots of problems before achieve the final clinical application including the classification and definition of EVs which still have consensus about it.In April 2012 Sweden international conference on microvesicles was held in Gothenburg.The host was emphasizing the importance of naming and classification with EVs.In the conference has reached some consensus including in EVs are not completely defined and named.So the conference suggested that we should use EVs to replace the name of different kinds of vesicles such as exsome.Most of urine specimens used in current researches about uEVs are the morning or random urine samples,due to the limitation of the separation method for uEVs.But lots of reports suggestted that there was great difference between the uEVs amount of diffentent individuals for a single urine sample,and there was also circadian rhythm for uEVs secretion.Therefore it is very necessary to analyze the dynamic secretion of uEVs within 24 hours which is helpful for deeply understanding the secretion status of uEVs in healthy adults.Untill now there is no report in the field.During the study of the biomarkers for disease diagnosis in the past 10 years.there was rapid development in the developlent and optimization of proteomic technology.Urine can be easily used in clinical and basic researches.as it is of advantages of simple.non-invasive.adequate and stable.Based on the researches of uEVs.researchers can well overcome the disadvantages of routine urine samples auch as the miscellaneous of contents.poor stability.great deviation of the results.But it is still lack of report about the difference of protein contents in urine vesicls between different genders by means of proteomic analysis.It has been reported by a large number of studies that there is significant gender difference between the incidences of diseases?as well as the gender difference between either the fingerprint or the quantity of proteins in the urine.Thus,for studying disease biomarkers?it is reasonable to initially understand the gender difference of urine proteins.It is useful for well analyze and compare the difference of protein expression between diseases.However there is lack of the reports about the difference of urine vesicle proteins between denders by means of proteomic analysis.Our present study successed in the extraction and analysis of uEVs from the 24-hour urine of healthy individuals by means of hydraulic dialysis method whch was recently reported by our goup.It is the first report about the content and size distribution of 24-hour urine uEVs in healthy adults and the differences between individuals.Based on the step by step trails of our group in recent 3 years,we successed in urine uEVs enrichment and purification with our new uEVs proteomic technique,hydraulic dialysis-reduction-alkylation-Trypsin digestion followed by mass spectrometry.For the first time we determined the difference of uEVs internal and external proteins,and for the first time,analyzed the gerder difference of the protein fingerprint and contents of uEVs in 24-hour urine samplesMaterials and methods1.1 Urine collectionUrine was collected from nine healthy adults(aged 22 to 63,the median was 49).The blood glucose,fasting insulin,routine urine,24 hours urine protein quantitative,24 hours urine trace albumin,serum creatinine detection and evaluation of eGFR were be examined to rule out any chronic disease such as chronic kidney disease and diabetes.Confirm all of healthy adults without an acute infection and the history of drug abuse,avoid menstrual period if female at lease 2 week before we collect the 24-hour urine.Our study use toluene which has been verified that will not influrnce with uEVs by our team as a preservative in urine.1.2 uEVs Isolation24-hour uEVs isolation with "Hydrostatic filtration dialysis"with specific steps as follows:(1)The sample was centrifuged at 2,00O×g for 30 min at room temperature to remove cells,cellular debris and partly of Tomm-Horsfall protein;(2)The 2,000×g supernatant was pour into dialysis filtration device with molecular weight cut-off(MWCO)of 1,000 kDa(3)Reach the volum of 6?8 mL in the dialysis membrane,add 200 mLmiliQ water to wash the sample;(4)After reaching the volum of 15?20 mL in the dialysis membrane and collecting the liquid inside of the membrane which is rich in uEVs,each separate uEVs samples top up to 20 mL with water.1.3 BCA(bicinchoninic acid)Prepare BCA standards.Prepare 1 ml of BCA stock(2 mg ml-1,dissolved in H20)and then make serial(5-8)dilutions with a range of 0?2mg/ml.as follows:0,0.025,0.125,0.25,0.5,0.75,1,1.5,2 mg/ml.Sample(sample BSA solution or protein solution)and PBS,a total of 20?L sample,finally plus 160?L working liquid blending for each hole,37? for 30 min incubation,after cooling to room temperature,enzyme 562 nm on the instrument readings.1.4 Western blot(WB).Preparing the gel with the concentration of 15%.Heat the sample to 100? for 15min before loading 30 ?L of protein sample into the wells of the SDS-PAGE gel.Covering the gel with 1×running buffer and run the gel with 20mA for 1?1.5h.The membrane can be either nitrocellulose or PVDF.Activate PVDF with methanol for 1 min and rinse with transfer buffer before preparing the stack.Blocking the membrane for overnight at 4? using blocking buffer.Incubate the membrane with appropriate dilutions of primary antibody(TSG101)in blocking buffer(dilution resistance,1:250),.Wash the membrane in three washes of TBST,5 min each.Incubate the membrane with the recommended dilution of conjugated secondary antibody(one fight for rabbit anti Tumor Susceptibility Gene101,TSG101).in blocking buffer at buffer 37 ? for 2 h.Wash the membrane in three washes of TBST,5 min each.Exposure to join liquid A and B(mixed in 1:1)each 1 mL.Remove the membrane to the dish,gently drain on filter paper,fixing,and scan.1.5 Transmission electron microscopy(TEM)Taking 20?30 uL uEVs precipitation suspension in copper net load samples,putting at room temperature for 2 min,using filter paper for sucking liquid from the side,adding about 30 uL of 4%phosphotungstic acid solution to copper net,negative dyeing 2 min at room temperature.After staining,samples were imaged by PHLIPS-TECNAI10 electron microscope.1.6 Tracking and analyzing the nanoparticles(Nanoparticle tracking analysis,NTA)NTA detectable in the range of 107?109 particles/mL,take healthy adult urine article(9),press 1:1000 scale with miliQ water diluted into I mL,via NanoSight 300(NanoSight,pennsylvania-based firm,UK)measurement,temperature of 23.7 ?.25 frames/s,measuring time for 60 s,using the principle of vesicle Brownian motion and provide sample visualization and approximate the function of the vesicle concentration.Eventually get the concentration and the size of the vesicles two-dimensional(2d)distribution.Optimize the NTA Settings and ensure the parameters the same when measuring different samples,the approximate analytical sample concentration,the distribution of different size vesicles subgroup.1.7 Urine vesicle purification and vesicle internal protein fingerprint analysis"Hydraulic dialysis method" was used for the concentration of uEVs,which was followed by reduction and alkylation reaction and Trypsin digestion reaction for uEVs purification.Then detergent sodium deoxycholic acid was used to bread the uter membrane ofuEVs for exposuring uEVs internal components,and then alkylation and Trypsin enzymic protein was used for restoring proteins.The inside protein fingerprints were determined by means of mass spectrum(MS).1.8 Statistical analysisSPSS 19.0 software was used to analyze the data.The measurement data were shown with meanąSD(standard deviation).The non-measurement data were shown with ratio.Individual difference was shown with coefficient of variation(CV).The calculating formula of coefficient of variation was standard deviation/average ×100%.Conclution1."Hydrostatic filtration dialysis" was used as the new uEVs separation and enrichment method for the first time for healthy adult 24-hour urine uEVs enrichment,separation,and the process of removing impurity.Our new method helps us to get enough uEVs and the enough quantity of uEVs is the basis for uEVs research.The new method is suitable for different labs,different people and different physiological and pathological state for uEVs study.The new method we established in the the study is worth popularizing widely.The application of the technology is of advantages such as simple equipment,easy to operate,the characteristics of high repeatability.2.The success of our study for the new method is of value in the extraction of 24-hour uEVs and the analysis for its secretion amount and size distribution in different individuals.It is shown by the results of our present study that,for healthy adults,there was only little diffetence between the 24-hour amount of uEVs secrestion of different individuals,It is suggested that 24-hour uEVs might be ideal specimen for uEVs related research.Conform to the scope of micro vesicles in 100?1000 nm in diameter of vesicle accounted for the main part of the human body secretion uEVs.3.By means of the 24-hour uEVs proteomic analysis,we identified reliable uEVs internal protein map of healthy acults.And on the basis of the comparison,it is found that there was differetnce between the protein fingerprints in the uEVs of different genders.It is suggested that,in the research of the proteomic analysis of urine extracellular vesicles,we should pay attention to the different expression of protein which maybe caused by gender differences. |
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