| Objective:To study the number,morphological and molecular differences of exosomes in pleural effusion with three different causes,that is,to analyze the similarities and differences in the characterization of exosomes in pleural effusion with different causes.Search for potential tools that can quickly and accurately identify the cause of pleural effusion.Method:Collect pleural effusion with three different causes: malignant(lung adenocarcin noma),tuberculous and leaky pleural effusion.Exo View detection kit was used to capture exosome containing specific protein markers by SP-IRIS and immune capture anti bodies,and the number and particle size of exosome in pleural effusion with different causes were directly detected.The phenotype of individual exosomes was detected by labeling exosome surface proteins with fluorescent antibodies.Fluorescen ce signals of each exosome in pleural effusion of different causes were detected and statistically analyzed to obtain fluorescence co-localization information,and then similarities and differences in characterization analysis of pleural effusion of three different causes were compared.Results:1.Enumeration and distribution analysis of exosome subgroups in different causes of pleural effusion: The number of exosomes containing CD63+ or CD81+transmembrane protein was the highest in pleural effusion with three different causes,and the number of exosomes containing CD9+ transmembrane protein was the lowest,but all of them were significantly higher than MIg G negative control.There was no significantly statistical difference in the number of exosomes containing CD63+,CD81+ and CD9+ transmembrane proteins in pleural effusion of three groups with different causes(P>0.05).Proportion of exosomes containing CD9+ transmembrane proteins in tuberculous pleural effusion(B1:12.45%;B2:18.77%;B3:19.96%)were lower than those of lung adenocarcinoma pleural effusion(A3:28.58%;A5:20.15%)and leakage(C1:22.19%).In adenocarcinoma pleural effusion of lung,the number of exosomes in A5 samples was significantly higher than that in A3 samples.The percentages of CD63+ and CD9+ exosome subgroups in A5 samples(37.07% and20.15%)were lower than those in A3 samples(40.49% and 28.58%),while the percentages of CD81+ exosome subgroups in A5 samples were higher than those in A3samples(42.78% VS 31.03%).In tuberculous pleural effusion,the number of exosomes in B1 samples was significantly higher than that in B2 and B3 patients,and the proportion of CD63+ exosome subgroup in B1(49.35%)was higher than that in B2 and B3(40.17% and 34.29%).The percentages of CD9+ and CD81+ exosome subgroups in B1(12.45%,38.20%)were lower than those in B2(18.77%,41.06%)and B3(19.96%,46.38%).2.The exosome particle size containing different transmembrane proteins in pleural effusion with different causes: In lung adenocarcinoma and tuberculous pleural effusion,the mean particle size of exosomes containing CD81+ transmembrane protein was the largest,followed by those containing CD9+ transmembrane protein,and those containing CD63+ transmembrane protein were the smallest.In leaky pleural effusion,the mean particle sizes of exosome subgroups containing CD63+,CD81+ and CD9+were the same,all of which were 57 nm.However,there was no statistical difference in exosome particle size containing CD63+,CD81+ and CD9+ transmembrane proteins in pleural effusion of different properties among the three groups(P>0.05).3.Comparison of the number of fluorescent antibodies in pleural effusion with different causes in different antibody capture channels: In the CD63+ antibody capture channel,the number of CD63 fluorescent antibodies was the highest in both lung adenocarcinoma pleural effusion and tuberculous pleural effusion,and the number of CD81 fluorescent antibodies was the highest in the leaked effusion.The number of CD9 fluorescent antibodies was the highest in lung adenocarcinoma pleural effusion and leaked effusion in the CD81+ antibody capture channel.The number of CD81 fluorescent antibodies was the highest in tuberculous pleural effusion and leaked effusion in CD9+ antibody capture channel.In lung adenocarcinoma pleural effusion,the number of exosomes labeled with CD63 fluorescent antibodies was the largest in CD63+ antibody capture channel,and the number of CD9 fluorescent antibodies was the largest in CD81+ antibody capture channel,and the number of other exosome fluorescent antibodies was not the same.In tuberculous pleural effusion,B1,B2 and B3 had similar distribution of fluorescent antibodies in the CD63+ capture channel,with CD63 having the highest number of fluorescent antibodies,followed by CD81 and CD9 having the least.In CD81+ and CD9+,antibody capture channel,B2 and B3 showed similar trends in the number distribution of fluorescent antibodies.In both CD81+ channels,the number of exosomes labeled with CD81 fluorescent antibodies was the highest,followed by CD63,and the number of exosomes labeled with CD81 fluorescent antibodies was the lowest.There was no significant difference in the number of exosomes labeled with CD63 and CD9 fluorescent antibodies,but the number of exosomes labeled with CD9 fluorescent antibodies was slightly more than that labeled with CD63 fluorescent antibodies.The distribution of exosomes in patients with B1 was significantly different from that in patients with B2 and B3.In the CD81+antibody capture channel,the number of exosomes labeled by CD63 fluorescent antibody was the largest,followed by CD81 fluorescent antibody and CD9 fluorescent antibody.In the CD9+ antibody capture channel,the number of exosomes labeled by CD81 fluorescent antibody was the largest,followed by CD63 and CD9.4.Colocalization analysis of exosomes with different causes of pleural effusion: In exosomes of mono yang,CD63 accounted for the highest proportion of pleural effusion of three different causes(A3:43.2%,A5:41.4%,B1:68.7%,B2:52%,B3:37.6%,C1:20%),and CD81 followed in tuberculous pleural effusion(B1:17.4%,B2: 28.7%,B3:25.3%),the least CD9(B1:10.5%,B2:14.9%,B3:12.8%).In CD63+ subgroup,CD81+ subgroup and CD9+ subgroup,CD63/81/9 exosomes accounted for the highest proportion(38.7%,35.6%,49.4%,respectively)in leaky pleural effusion.In the three cases of tuberculous pleural effusion,the proportion of exosomes CD63/81 in CD63+subgroup of Shuang yang was higher than that in CD63/9(B1:18.5% VS 6.1%,B2:28.9%VS 6.7%,B3:42.9% VS 7.7%).The proportion of exosomes CD63/81 in CD81+subgroup was higher than that in CD81/9(B1:40.3% VS 19.5%,B2:33.7% VS 18.6%,B3:35.3% VS 23.1%).In CD9 + subgroup,the proportion of exosome CD9/81 in Shuang yang was higher than that in CD9/63(B1:35.2% VS 11.0%,B2:34.8% VS10.3%,B3:43.0% VS 14.0%).Conclusion:1.There was no statistically significant difference in the number of exosomes containing CD63+,CD81+ and CD9+ transmembrane proteins in different causes of pleural effusion,but the distribution of exosome subsets in different individuals with different or the same cause of pleural effusion was different.The proportion of exosomes containing CD9+ transmembrane proteins in tuberculous pleurisy was lower than that in lung adenocarcinoma and transutural pleurisy.2.There was no statistically significant difference in the particle size of exosomes of different transmembrane protein subsets in different causes of pleural effusion.However,the average particle size of exosomes containing different transmembrane proteins in lung adenocarcinoma pleural effusion and tuberculous pleural effusion were different,and the average particle size of exosomes containing CD81+ transmembrane protein was the largest in both these pleural effusion.The average particle size of the subsets of exosomes containing three different transmembrane proteins in leaky pleural effusion was the same,all of which were 57 nm.3.There are differences in the distribution of different fluorescent antibodies in different antibody capture channels between pleural effusions of different causes and pleural effusions from different individuals with the same cause.4.There are exosomes expressing different transmembrane proteins in pleural effusion of different causes,and one two or three of the three transmembrane protein markers CD63,CD81 and CD9 could be present,and the proportion was also different,which indicated that the co-localization analysis of exosomes in different causes of pleural effusion was heterogeneous.This may serve as a potential marker for the abductive cause of pleural effusion. |
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