不同来源外泌体对肝癌生长及转移的影响研究  

Effects of exosomes from different sources on the growth and metastasis of hepatocellular carcinoma

作　　者：齐寒 张缘 左冰峰 刘志礼 吴丽 尹海芳 Qi Han;Zhang Yuan;Zuo Bingfeng;Liu Zhili;Wu Li;Yin Haifang(School of Basic Medical Sciences, Tianjin Medical UniversitT, Tianjin 300070, Chin)

机构地区：[1]天津医科大学基础医学院,300070

出　　处：《国际生物医学工程杂志》2018年第2期148-153,159,共7页International Journal of Biomedical Engineering

基　　金：天津医科大学科学基金（2014KYQ20）

摘　　要：目的 在肝癌原位移植瘤小鼠模型上系统评估不同来源的外泌体对肝癌生长、转移及免疫微环境的影响,为肝癌临床免疫治疗提供新的思路.方法 采用超速离心法分别获取小鼠肝癌细胞Hepa1-6及肝癌原位移植瘤小鼠（接种原位瘤后3周）血清来源的外泌体,并通过透射电子显微镜鉴定外泌体的形态.将9只接种原位瘤1周后的C57BL/6小鼠随机分为3组（每组3只）,分别尾静脉注射120μl PBS（对照组）、Hepa1-6细胞来源的外泌体（1μg/μl）（TEXcel组）、肝癌原位移植瘤小鼠血清来源的外泌体（1μg/μl）（TEXserum组）,每周注射1次,连续注射3周.末次注射后第4天处死小鼠,剖取肝癌组织和肺组织,测量肝癌组织的体积;蛋白质印迹法（Western Blot）检测肝癌组织中程序性细胞死亡蛋白-1配体（PD-L1）的表达;免疫组化染色检测肝癌组织中CD3＋、FoxP3＋T淋巴细胞的浸润情况;苏木素-伊红染色检测肝癌肺转移情况.结果 Hepal-6细胞来源的外泌体和肝癌原位移植瘤小鼠血清来源的外泌体直径均在100 nm左右,呈均一的囊泡状,且具有完整的膜结构.与对照组相比,Hepa1-6细胞来源的外泌体对肝癌生长及肺转移具有一定的抑制作用;而TEXserum组与对照组无明显区别.Western Blot检测结果显示,与对照组相比,TEXcel组和TEXserum组肝癌组织中的PD-L1蛋白表达均明显上调.免疫组化染色结果显示,与对照组相比,TEXcel组和TEXserum组肝癌组织中FoxP3＋标记的调节性T细胞（Treg）浸润数无明显变化,差异均无统计学意义（均P〉0.05）;但CD3＋标记的T淋巴细胞浸润数明显减少,差异均具有统计学意义（均P〈0.05）.结论 Hepa1-6细胞来源的外泌体对肝癌生长和肺转移具有一定的抑制作用,但对肝癌免疫微环境无明显调控作用;而肝癌原位移植瘤小鼠血清来源的外泌体能促进肝癌生长、肺转移及免疫抑制微环境.Hepa1-6细胞来源的Objective To evaluate the effect of exosomes from different sources on the growth, metastasis and immune microenvironment of hepatocellular carcinoma （ HCC ） in a mouse model of orthotopic transplanted hepatoma so as to provide new insight into the clinical immunotherapy of hepatocellular carcinoma. Methods The serum-derived exosomes were obtained by ultracentrifugation from Hepa1-6 cells and 3-week orthotopically tumor-bearing HCC mice. The morphology and size of exosomes were identified by transmission electron microscopy. One-week tumor-bearing HCC C57BL/6 mice were randomly divided into 3 groups （ 3 mice for each group ） , and respectively treated with tail vein injection of 120 μl PBS（control group）, 120 μl Hepa1-6 cell-derived exosomes （1μg/μl, TEXcel group）, and serum-derived exosomes （1μg/μl） （TEXserum group）. The treatments were conduced once a week for 3 consecutive weeks. The mice were sacrificed on the 4th day after the treatments, and the liver tissue and lung tissues were dissected. The volumes of the liver cancer tissues were measured. The expression of PD-L1 protein and CD3＋and FoxP3＋T lymphocytes infiltration in the liver cancer tissues were respectively detected by Western Blot and immunohistochemical staining. The lung metastasis of the liver cancer was detected by hematoxylin-eosin staining. Results The diameters of the Hepa1-6 cell- and serum-derived exosomes both were about 100 nm, and were uniform vesicles with complete membrane structure. Compared with the control group, Hepa1-6 cell-derived exosomes had a certain inhibitory effect on the growth and lung metastasis of HCC, while there was no significant difference for the TEXserum group. Western Blot results showed that compared with the control group, PD-L1 protein expression was significantly up-regulated in both TEXcel group and TEXserum group. Immunohistochemical staining＆nbsp;showed that compared with the control group, the infiltration number of Foxp3＋-labeled regulatory T cells （Treg） had no

关 键 词：外泌体 肝癌 生长 转移 免疫微环境 

分 类 号：R735.7[医药卫生—肿瘤]