Research On Isolation Methods Of Urinary Exosome

Objective To compare the separation effect of exosome derived from human urine by three methods include in ultracentrifugation,ultrafitration and precipitation. And futher to explore the optimal conditions for urinary exosome separation. This study provides the referential basis for clinically chosing appropriate method to separate human urinary exosome.Methods1. The urinary exosome in 20 healthy volunteers and 10 patients with chronic kidney disease(CKD) were separated and extracted respectively by ultracentrifugation, ultrafiltration and precipitation. To identify the exosome, the size and shape of exosome was observated by transmission electron microscopy(TEM) and the expression of makers TSG101 and Alix in exosome was tested by Western-blot.2. In order to compare the separation effect of exosome separated by3 different methods, the marker proteins in urinary exosome were detected by Western-blot in the conditions of the same urine volume and equal total protein in exosome.3. The maker proteins in exosome were tested by Western-blot in order to analyze weather it was nessary to remove cells by low-speed centrifugation before 17,000 g centrifugation and analyze the influence of different centrifugal force(100,000 g, 150,000 g, 200,000 g and 250,000g),centrifugal time(0.5h, 1h and 2h) and temperature(4℃, 25℃ and 37℃)on separating urinary exosome by ultracentrifugation.Results1. As observed under TEM, exosome separated by 3 methods was round or oval vesicle with integrated membrane, 40-100 nm in diameter and in which had uniform low electron density contents. The results of marker preoteins TSG101 and Alix in exosome detected by Western-blot showed that both two makers TSG101 and Alix were detectable in exosome separated by ultracentrifugation and ultrafiltration, while they were both not detectable in the supernatant of ultracentrifugation, the filtrate of ultrafiltration, the precipitate and supernatant of precipitation.2. In the condition of the same urine volume, the marker proteins TSG101 and Alix in exosome separated by 3 methods were detected by Western-blot. The results showed that markers in exosome separated by ultracentrifugation had the darkest band, and both markers could be detected in all specimens. Exosome separated by ultrafiltration, TSG101 and Alix protein could be detected in exosome derived from healthy volunteers urine, but not in exosome derived from CKD patients. And both markers in exosome separated by precipitation could not be detected in all samples. Adjusting the sample volume to obtain the equal exosome total protein content, the marker proteins TSG101 and Alix in exosome separated by 3 methods were detected by Western-blot. The quantity of exosome separated by ultracentrifugation was the most and in which TSG101 and Alix could be detected in all samples. Exosome separated by ultrafiltration could detect TSG101 and Alix in exosome derived from 11 out of 20 healthy volunteers, but could not detected in exosome derived from all CKD patients. And markers in exosome separated by precipitation could not be detected in all samples.3. According to the results of Western-blot, there was no obviously difference in the band gray level of marker proteins in exosome separated from urine samples removed cells by low speed centrifugation or not. The band gray level of marker proteins in exosome was darker with increased centrifugal force or centrifugal time and was the darkest in the condition of 250,000 g,2h, but had no significantly difference under different temperature conditions.Conclusions Compared to the ultrafiltration and precipitation,ultracentrifugation had the highest efficiency to separate the urinary exosome which was deserved to be recommend. It was unnecessary to remove cells by low-speed centrifugation before 17,000 g centrifugation. Separation effect of urinary exosome by ultracentrifugation was the best on the conditions that the centrifugal force was 250,000 g and the centrifugal time was 2h. the centrifugal temperature had no significantly influence on urinary exosome separation.