| Objective:To build a novel efficient exosome isolation chip based on electroosmotic pump,and to characterize the exosomes extracted by the chip;to analyse the expression difference of ExoPD-L1 in patients with NSCLC,patients with benign lung diseases and healthy people,and to explore the application value of plasma ExoPD-L1 in the diagnosis of NSCLC.Methods:The research participants were 91,including 45 patients with NSCLC,16 patients with benign lung diseases and 30 healthy people.1.An integrated exosome isolation chip based on electroosmotic pump chip and gel electrophoresis microfluidic chip was developed by using nanopore membrane.Plasma exosome was extracted by this mentioned method.The size and shape of exosome was observed by TEM.The concentration and particle size distribution of exosome was detected by Nanosight.And the expression level of marker protein,such as HSP70 and TSG101 on exosome surface,was detected by Western blot.2.The plasma exosome of participants was isolated by the mentioned exosome isolation chip,and the concentration of ExoPD-L1 was detected by ELISA.The expression difference of ExoPD-L1 among NSCLC group,benign lung diseases group and healthy control group was compared.The relationship between ExoPD-L1abundance and clinical characteristics of NSCLC patients was analyzed.With the pathological biopsy as the gold standard,the diagnosis difference between ExoPD-L1expression level and“five lung cancer markers”was compared.Results:1.The exosome isolation chip based on electroosmotic pump chip was successfully established.The morphology of exosome,which is complete,saucer-like,and about 100nm in diameter,was confirmed by TEM.The results of Nanosight showed that the concentration of exosomes was about(1.13±0.08)×10~9/mL and the diameter of exosomes was concentrated in 50-150 nm.The marker protein HSP70 and TSG101 on exosome surface were detected by Western blot.2.The content of ExoPD-L1 in NSCLC group,benign lung diseases group and healthy control group was statistically described in the form of(median,quartile range),they were(15.77 pg/mL,16.76 pg/mL),(8.37 pg/mL,3.13 pg/mL),(5.54 pg/mL,5.65pg/mL).Kruskal Wallis H test showed that the expression level of ExoPD-L1 in three groups was not same(H=38.229,P<0.01),which in NSCLC group was obviously higher than other groups.Pairwise comparison analysis showed that the expression of ExoPD-L1 in NSCLC group was different with benign lung diseases group and healthy control group(P<0.01),but no difference was detected between benign lung diseases group and healthy control group(P>0.05).3.The expression of ExoPD-L1 in NSCLC patients was correlated with tumor stage,lymphatic metastasis,distant organ metastasis and clinical stage(P<0.05).The higher tumor stage(T3/T4 vs.T1/T2,P=0.038),lymphatic metastasis(N1/N2 vs.N0,P=0.003),distant organ metastasis(M1/M2 vs.M0,P=0.013)and later clinical stage(Ⅲ/Ⅳ vs.Ⅰ/Ⅱ,P=0.003)mean higher expression level of ExoPD-L1.The expression level of ExoPD-L1 in NSCLC patients was not correlated with age,gender and smoking(P>0.05).4.With pathological biopsy as the gold standard,the expression level of ExoPD-L1was used to diagnosis NSCLC,and the ROC curve was drawn.The diagnostic cut-off value was 9.65 pg/mL(95%CI:0.823-0.961).The sensitivity and specificity of the method were 77.8%and 86.7%,P<0.01.Usingthe ExoPD-L1 method and“five lung cancer markers”method to diagnosis the patients with lung neoplasms,respectively,the results showed that the sensitivity of ExoPD-L1 method was 77.8%(35/45),specificity was 81.3%(13/16),the sensitivity of“five lung cancer markers”method was 46.7%(21/45),specificity was 87.5%(14/16);The consistency was analysed between two methods and the pathological biopsy,the kappa value was 0.517 and 0.237,respectively;Mc Nemar’s test suggested that there was significant difference between the two methods in the diagnosis of NSCLC,P<0.01.Conclusions:1.The exosome isolation chip based on electroosmotic pump chip can successfully isolate exosomes with complete shape and satisfactory function from plasma samples,which can be used in subsequent experiments.2.The expression level of ExoPD-L1 in NSCLC patients was apparently higher than that in benign lung diseases group and healthy control group,and the concentration was positively correlated with tumor stage,lymphatic metastasis,distant organ metastasis and clinical stage.The plasma ExoPD-L1 diagnosis method had satisfactory sensitivity and specificity,and better consistency with the gold standard than the“five lung cancer markers”method.Therefore,ExoPD-L1 could be a promising factor on tumor screening and auxiliary diagnosis,has good application value in clinical diagnosis of NSCLC. |
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