结直肠癌患者血浆外泌体sHLA-G表达及临床意义

Expression and clinical significance of sHLA-G in plasma exosome in patients with colorectal cancer

目的检测结直肠癌(CRC)患者血浆外泌体可溶性HLA-G(sHLA-G)水平并评估其临床价值。方法回顾性研究。选取2017年5月至2018年8月浙江省台州医院收治的原发性CRC患者52例,同时选取25名健康体检者,20例良性结直肠息肉患者,20例慢性肠道炎症作为对照。采用exoEasyMaxi试剂盒提取血浆外泌体,纳米颗粒示踪技术(NTA)和免疫印迹法用于鉴定外泌体。流式细胞术(FCM)和酶联免疫吸附法(ELISA)检测外泌体sHLA-G水平,并用ROC曲线和约登指数(Youdenindex)分析其与癌胚抗原(CEA)、糖类抗原19-9(CA19-9)在CRC诊断中的差异。结果CRC血浆提取的外泌体颗粒大小峰值为101.1nm,表达CD63、CD81和TSG101蛋白。CRC患者外泌体sHLA-G[28.0(21.5~35.1)U/ml]显著高于良性结直肠息肉患者[19.7(16.2~22.5)U/ml,U=180.0,P<0.001]、肠道慢性炎症[19.9(16.7~25.2)U/ml,U=197.0,P<0.001]和健康对照组[19.6(16.8~21.3)U/ml,U=143.0,P<0.001],且术后sHLA-G[19.6(17.8~26.3)U/ml,325.5,P=0.015]显著低于术前水平。CRC患者外泌体sHLA-G在肿瘤分期(U=64.0,P=0.006)、淋巴结转移(U=81.0,P=0.003)及TNM分期(U=105.0,P=0.015)等分组中存在显著性差异。ROC曲线分析表明,FCM和ELISA方法测定外泌体sHLA-G、CEA、CA19-9的曲线下面积(AUC)分别为0.962±0.019、0.899±0.038、0.786±0.058、0.680±0.068。AUC经Z检验表明:在诊断CRC效能上,FCM测定外泌体sHLA-G优于CEA(Z=2.884,P=0.004)和CA19-9(Z=3.994,P<0.001),ELISA测定外泌体sHLA-G优于CA19-9(Z=2.811,P=0.005)。结论血浆外泌体sHLA-G与CRC患者的疾病进展相关,且在诊断效能上优于传统肿瘤标记物CEA和CA19-9。

Objective To detect sHLA-G expression in plasma exosomes in patients with colorectal cancer and evaluate its clinical significance. Methods Retrospective study.Plasma was collected from 52 primary CRC patients,20 colorectal polyps patients, 20 inflammatory bowel disease patients and 25 healthy donors in the Taizhou Hospital of Zhejiang Province from May 2017 to August 2018. The exosomes were extracted by exoEasyMaxikit and identified by nanoparticle tracking analysis (NTA) and Western blot. Exosomal sHLA-G was detected by flow cytometry (FCM) and enzyme-linked immunosorbent assay (ELISA). The diagnostic values of exosomal sHLA-G detected by FCM and ELISA were assessed,and their diagnostic performances were compared with carcinoembryonic antigen (CEA) and carbohydrate antigen CA19-9 by ROC curve and Youden index. Results The peak size of exosomes extracted from plasma in CRC patients was 101.1 nm and Western blot showed these exosomes expressed marker CD63, CD81, and TSG101.Exosomal sHLA-G of CRC patients [28.0(21.5-35.1)U/ml] was significantly higher than that in healthy controls[19.6(16.8-21.3) U/ml, U=143.0, P<0.001],colorectal polyps patients[19.7(16.2-22.5)U/ml, U=180.0, P<0.001] as well as inflammatory bowel disease patients[19.9(16.7-25.2)U/ml, U=197, P<0.001].The postoperative sHLA-G level[19.6(17.8-26.3)U/ml, U=325.5, P=0.015] was significantly lower than that in pre-operation. Exosomal sHLA-G was significantly different in different tumor status(U=64.0, P=0.006), lymph node metastasis (U=81.0, P=0.003) and TNM stage (U=105.0, P=0.015) in patients with CRC. ROC curve showed the area under the curve (AUC) of exosomal sHLA-G detected by FCM and ELISA, CEA and CA19-9 was 0.962±0.019,0.899±0.038,0.786±0.058, 0.680±0.068, respectively. The difference of AUC was operated by Z test, and it showed that the exosomal sHLA-G detected by FCM was superior to CEA(Z=2.884, P=0.004)and CA19-9(Z=3.994, P<0.001), and the exosomal sHLA-G detected by ELISA was superior to CA19-9(Z=2.811, P=0.005). Conclusion Plasma exosomal sHLA-G was associated with the progression of CRC and its diagnostic value was superior to the traditional tumor markers CEA and CA19-9.