| The incidence rate of prostate cancer in China is increasing year by year,which seriously endanger the health of male citizens in China.The diagnosis of prostate cancer mainly depends on pathological biopsy,but it is invasive.Low serum PSA specificity.Therefore,it is necessary to continue to search for new non-invasive or minimally invasive detection methods and markers for the diagnosis of prostate cancer.Exosomes are new stars in the field of liquid biopsy,reflecting the pathophysiological state of the source cells.As a new marker for disease diagnosis,it has great potential.Studies have confirmed that PSMA and P504 s are present in serum and are of great value in the diagnosis of prostate cancer.Based on this,the expression of PSA,PSMA and P504 s in exosomes and their relationship with prostate cancer are explored,We conduct the following three parts of research.Part 1 Objective:Establishing a method for enriching exosomes by one-step method of carboxyl magnetic bead-conjugated antibody to study the value of prostate cancer related exosomes in the diagnosis of prostate cancer.Methods:The double-antibody sandwich principle was used to optimize the key parameters in the enzyme-free and magnetic bead system.The magnetic bead system was used to capture the prostate-related exosomes in the serum,and the exosome was analyzed in prostate cancer,benign prostatic hyperplasia,and healthy controls.The expression was confirmed by electron microscopy and Western blot.Results:Screening blocking solution 1 as the best blocking solution.The sample loading was70 ul and the sample incubation time was 2h.The enzyme concentration of1:2000 were selected.In magnetic bead system,the antibody volume is 15 ul,the coating liquid has a p H of 5.5,and 1.03 um Merck carboxyl beads as the best condition.It is not concluded that the coating efficiency of multipleantibody-coated magnetic beads and the captured exosomes must be higher than that of the single antibody,but are related to the antibody.As prostate cancer progressed,the number of exosomes released increased.The expression of PSA and PSMA was different between prostate cancer and healthy controls.It was proved that PSA and PSMA included in the exosome,and significant correlation between PSA in serum and PSA in exosomes.Transmission electron microscopy showed that the size of the vesicles captured by the one-step technique of carboxyl-bead-conjugated antibody was between 30-100 nm,which was consistent with the size of the exosomes.Western blot was used to detect the expression of exosome universal protein and prostate cancer-associated exosomal proteins.Conclusion:It is believed that the one-step technique of carboxyl-bead-conjugated antibody constructed and optimized in this experiment was successfully used to detect exosome surface proteins and is applied to the detection of prostate cancer related exosomes.Part 2 and Part 3 Objective:To explore the effects of several methods used by researchers to extract serum and urine exosomes,and further confirm the feasibility of the carboxyl magnetic beads in this experiment to capture serum and urine exosomes.It provides a basis for the detection of prostate cancer,an easy-to-collect,large-volume,non-invasive ideal body fluid for urine.Methods:Serum and urine exosomes of prostate cancer patients were enriched by ultracentrifugation,commercial kits and magnetic beads.The exosomes were confirmed by electron microscopy and Western blot.Results:Electron microscopy showed that vesicles with a size of30-100 nm were captured,and different methods captured the exosomes of different protein subpopulations.The comparison with ultracentrifugation confirmed the feasibility of one-step enrichment of exosomes by carboxyl-bead-conjugated antibodies in this experiment.Conclusion:Preliminary exploration of urine exosomes capture after one-step capture of serum exosomes by carboxyl magnetic beads-conjugated antibody.The feasibility of this experiment is confirmed by comparison with other methods.It is used as a foundation for the study of serum or even easy-to-use urine for the diagnosis of prostate cancer. |
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