Detection And Clinical Significance Of Exosome And MicroRNAs In The Urine Of Bladder Cancer Patients

ObjectiveTo analyze the feasibility of a way of isolating exosome and microRNA(miRNA) from the urine of patients with bladder cancer by differential ultracentrifugation. Then perform the identification of exosome and miRNAs, and explore the clinical value of the two kinds of biomarkers. Methods1?Isolating exosome by differential ultracentrifugation1.1 Preparation of urine specimen. Firstly, retrieve the medical record database, then do statistic induction of the quantitative distribution of data from bladder carcinoma. Make an optimal strategy to collect the urine specimen.1.2. Differential ultracentrifugation. Divide the urine specimens into two groups. Do precooling of the centrifugal machine and rotors. Add some protective material into the urine, and then start. First wipe off cell debris at 2000 g, 10 minutes. Second wipe off larger extracellular vesicles at 17000 g, 45 minutes. Third at 200000 g, 70 minutes. Then resuspend the sediment using PBS, and centrifuge again. Last, add 40 ?l ddH2 O, and save it at-80?, or do the next test.2?The identification of exosome2.1. Transmission electron microscopy(TEM). Dilute 10 ?l exosome sample, and then do ultrasonic dispersion uniformity. Add sample to copper mesh, waiting for one minute. Then add 5 ?l paraformaldehyde, waiting for 10 minutes; PBS for 5 minutes, 3 times. Then glutaraldehyde solution for 10 minutes; PBS for 5 minutes, 3 times. Then 5 ?l phosphotungstic acid dye, for one minute. After dring it, do examination.2.2. Nanoparticle tracking analysis(NTA). Start the machine and set the parameter; add 3 ?l of the extract to 0.3 ml of water; inject the mixture into the equipment and start recording.2.3. Western blot. Do determination protein concentration in extracted exosome with BCA Protein Quantitation Kit. Do protein denaturation at 95 ?,5 minutes. Then protein electrophoresis at 120 V, protein transfer at 350 mA for 70 minutes. Do occluding on PVDF film. Do incubation of first antibodies for 2 hours, second antibodies for one hour at room temperature. Observe the effect of venography.3?The identification of mi RNA3.1 The extraction of miRNA. Do extraction of miRNA with miRNA Extraction Kit, and detect the density of mi RNA samples. Do dry purification of miRNA.3.2 Real-time quantitative polymerase chain reaction(qPCR). Last do qPCR. Melting temperature is 94 ?, time is 2 minutes. Denaturation temperature is 94 ?, time is 20 seconds. Annealing temperature is 62 ?, time is 34 seconds. Do cycling 43 times. Results1. Urine samples is easy to obtain and signing informed consent helps the high success rate of obtaining urine samples with 23/25.2. Differential centrifugation can isolate exosome from urine.3. The ressults of TEM?NTA and western blot showed lots of exosomes in the the extract. The sediment contains some extracellular vesicles.4. qPCR ressults showed differential expression of miRNA 15b-5p and miRNA 15a-5p in the urine of bladder cancer patients and healthy people. Conclusion1. We can cellect urine specimen from bladdr cancer patient easily.2. We can cofirmed lots of exosomes in the cellected urine specimen.3. It is credible that miRNA can be extracted from exosome.4. It is a stable and reliable technology to extract exosome and miRNA from urine of patient with bladder carcinoma by differential ultracentrifugation, and the urine will be a nice source of biomarkers to the diagnosis of bladder cancer in the nearly future.