外周血中外泌体检测在喉癌下咽癌早期诊断中的临床研究

Clinical research of exosomes in peripheral blood from patients with laryngeal carcinoma and hypopharyngeal carcinoma

目的提取并检测喉癌、下咽癌的患者外周血中外泌体(exosomes)并探讨其临床意义。方法收集我院2015年6月～2017年6月共计22例头颈肿瘤患者术前外周血,其中喉癌11例,下咽癌11例,所有患者均术后病理明确诊断,并收集11例正常人血液作为对照组,用试剂盒System Biosciences(EXOTC10A-1)提取exosomes。采用透射电子显微镜下观察exosomes形态,纳米粒子跟踪分析(NTA)对exosomes进行计数(即颗粒浓度)和测粒径,蛋白浓度定量试剂盒(BCA)检测exosomes蛋白浓度,蛋白质免疫印迹(WB)检测蛋白的表达。结果喉癌组和下咽癌组的上清液中可以分离出肿瘤来源的exosomes,透射电子显微镜下观察具有杯状结构的特征,直径在30～100 nm,其体积比对照组更大。喉癌组及下咽癌组的exosomes蛋白浓度与对照组相比显著增加。三组exosomes中均检测到CD63蛋白及CD81蛋白,未检测到Calnexin蛋白,exosomes颗粒浓度各组间无明显差异。与对照组比较,喉癌组exosomes粒径增加但无显著差异,下咽癌组的exosomes粒径显著增加。结论喉癌、下咽癌患者外周血清来源的exosomes是十分有潜力的肿瘤标志物,可能对喉癌、下咽癌的早期诊断带来帮助。

OBJECTIVE To study the clinical significance of exosomes in peripheral blood from patients with laryngeal and hypopharyngeal carcinoma. METHODS A total of 22 cases with head and neck cancer,including11 cases of lar yngeal cancer patients and 11 cases of hypopharyngeal cancer,had been collected. Meanwhile 11 additional normal subjects also had been collected as control. The exosomes were extracted from the collected bloods of the patients and normal subjects by System Biosciences(EXOTC10 A-1). The shape of exosomes was observed through the transmission electron microscope. Nanoparticle Tracking Analysis(NTA) was used to count the numbers and measure the particle diameter of the exosomes. Bicinchoninic acid(BCA) was used to identify the protein concentration of exosomes. The expression of protein was detected by Western blot. RESULTS The exosomes can be separated out from the supernatant fluid of the blood from laryngeal and hypopharyngeal carcinoma patients. The separated exosomes under transmission electron microscope appeared as cup-shaped structure with a 30-100 nm diameter. The volume of exosomes from the blood of the patients was larger than that of the normal subjects. The protein concentration of exosomes in the blood of the patients was significantly higher than that of the normal control. The CD63 and CD81 proteins had been detected in the exosomes from the blood serums of normal subjects,laryngeal and hypopharyngeal carcinoma patients,while the Calnexin protein were failed to be detected. The particle concentrations of exosomes were not significantly different among the normal subjects,laryngeal cancer patients and hypopharyngeal carcer patients. Compared with the normal control,the particle diameter of exosomes from laryngeal cancer patients appeared larger,but with no evident difference between them. Meanwhile,the particle diameter of exosomes from hypopharyngeal cancer patients was significantly larger than that of normal control. CONCLUSION The exosomes from the blood serums of laryngeal and hypopharyngeal cancer patients have a great potential to be as the tumor marker,which it may be helpful to the early diagnosis.