口腔鳞癌细胞外泌体分离鉴定及基因表达谱研究

Isolation,identification and gene expression profile of exosomes from oral squamous cell carcinoma

目的口腔鳞状细胞癌（oral squamous cell carcinoma, OSCC）是口腔颌面部最常见的恶性肿瘤，易复发和转移，预后较差。寻找特异性的生物标志，进行无创性的早期筛查或诊断工具的开发，对改善OSCC患者预后差、生存率低、复发率高的现状至关重要。本研究旨在对OSCC细胞（Cal27）及口腔上皮细胞 （human oral epithelial cells, HOEC）来源的外泌体进行分离鉴定及基因表达谱分析，为寻找与OSCC相关的特异性外泌体提供研究基础。方法采用超速离心法从细胞培养上清中分离外泌体，透射电镜观察其形态特征，粒度仪分析其粒径，蛋白质印记法及流式细胞术检测其标志蛋白（ALIX、CD63、CD81）。利用高通量测序技术全面掌握两种外泌体基因表达谱，筛选差异表达基因，并进行生物通路分析。结果Cal27及HOEC外泌体均呈圆形或椭圆形，直径均分布在30～150Dill，ALIX、CD63、CD81标志蛋白呈现阳性表达。基因表达谱分析结果显示，两种外泌体间发生差异有统计学意义，｜log2Fold Changel〉｜，q〈0．001。表达变化的基因共有336个，KEGG生物通路富集分析发现共30个通路符合阈值要求（P〈0．05），其中32个基因（如ACVR1C、CDH20、THBS1等）及8个生物通路（如Wnt信号通路、P13K—Akt信号通路、TGF-8信号通路等）与癌症的发生发展密切相关。结论OSCC细胞及HOEC均能产生丰富的外泌体。0SCC细胞外泌体基因表达谱与HOEC外泌体基因表达谱相比存在很大的差别，通过对二者基因表达谱的分析，筛选出了可能与口腔鳞癌发生发展相关的差异表达基因及生物通路，为寻找与口腔鳞癌相关的特异性外泌体奠定了基础。

OBJECTIVE Oral squamous cell carcinoma（OSCC）,charactered with recurrence and metastasis,is the most common tumor in the oral and maxillofacial region. Detecting a special biomarker for non-invasive diagnostic and screening tool is very critical to improve the patients＇ status of poor prognosis,low survival rate and hign recurrence rate. This study aimed to isolate and identify the exosomes derived from oral squamous cell carcinoma cells（Ca127） and human oral epithelial cells（HOEC） and examine the exosomal gene expression profile,which can provide the foundation for detec- ting special exosome related to oral squamous cell carcinoma. METHODS Exosomes were isolated from cell culture super- natant by ultracentrifugation and further examined with transmission electron microscopy（TEM）, particle size analyzer, western blotting and flow cytometry. Using high-throughput sequencing technology to screen exosomal gene expression profiles for finding differently expressed genes,then creature information analysis were conducted to define gene functions and biological pathways. RESULTS The exosomes were round or oval in shape with a diameter between 30 to 150 nm, and they were positive in the expressions of ALIX,CD63 and CD81 proteins. A total of 336 genes showed differently ex- pressed（｜ log2Fold Change ｜〉1, q〈0. 001 ） and 30 biological ways met the requirement （P〈 0. 05） in the analysis, in which 32 genes （for example,ACVR1C,CDH20 and THBS1 ,et al. ） and 8 biological pathways （for example,Wnt signa- ling pathway,PI3K-Akt signaling pathway and TGF-β signaling pathway, et al. ） were closely related to the occurrence and development of cancer. CONCLUSIONS Abundant exosomes can be secreted by oral squamous cell carcinoma andhuman oral epithelial cells. The gene expression profile was obviously different between the two kinds of exosomes. The 32 differentially expressed genes and 8 biological pathways are associated with the biogenesis and development of oral squa- mous cell carcinoma,which provid study basis for detecting special exosome related to oral squamous cell carcinoma.