肝癌HepG2细胞来源外泌体通过环磷酸腺苷反应元件结合蛋白／叉状头／翅膀状螺旋转录因子3调控调节性T细胞的机制  

Hepatocellular carcinoma cell - derived exosomes regulates Treg cells via cyclic adenosine mono- phosphate response element binding protein/forkhead/winged helix transcription factor P3 pathway

作　　者：林泽伟[1] 程帝[2] 叶会霖 张光涛[1] 刘吉奎[1] 曾兵[3] Lin Zewei;Cheng Di;Ye Huilin;Zhang Guangtao;Liu Jikui;Zeng Bing(Department of Hepatobiliary Surgery, Peking University Shenzhen Hospital, Shenzhen 518035, China;Department of Hepatopancreatobiliary Surgery, Sun Yat - sen Memorial Hospital of Sun Yat -sen University, Shenzhen 510120, China;Department of Gastroin- testinal Surgery, the Sixth Affiliated Hospital of Guangzhou Medical University, Qingyuan 511518, Chin)

机构地区：[1]北京大学深圳医院肝胆外科,深圳518036 [2]中山大学孙逸仙纪念医院肝胆胰外科,广州510120 [3]广州医科大学附属第六医院胃肠外科,清远511518

出　　处：《中华实验外科杂志》2018年第6期1040-1042,共3页Chinese Journal of Experimental Surgery

基　　金：深圳市科技计划项目（JCYJ20150403091443331）;广东省自然科学基金（2015A030310099）;国家自然科学基金（81401996）

摘　　要：目的探讨肝癌HepG2细胞来源外泌体是否通过环磷酸腺苷（cAMP）反应元件结合蛋白／叉状头／翅膀状螺旋转录因子3（CREB／Foxp3）途径调控调节性T细胞（Treg细胞）。方法超速离心法分离外泌体（Exsomes）；透射电镜及Westernblot对外泌体进行鉴定；将HepG2和L02细胞来源外泌体分别与人外周血单个核细胞共培养，流式细胞检测Treg细胞比例的变化，Westernblot检测CREB／Foxp3信号蛋白的表达。结果见外泌体直径约30-100nm，呈圆形或椭圆形的囊泡状结构，且富含CD81和CD63蛋白。与L02-exo组[（1．87±0．18）％]和空白对照组[（1．43±0．12）％]比较，HepG2-exo组[（6．87±0．87）％]Treg细胞的比例显著上调，P值分别为0．005和0．004。HepG2-exo组CREB磷酸化分别是对照组和L02-exo组的1．74倍和1．63倍，P值均为0．001；HepG2．exo组Foxp3磷酸化分别是对照组和L02-exo组的1．93倍和1．78倍，P值均为0．001。结论肝癌细胞HepG2来源的外泌体可能通过CREB／Foxp3信号上调Treg细胞比例参与肿瘤免疫逃逸。Objective To investigate whether hepatocellular carcinoma cell line HepG2 derived exosomes regulates Treg cells via cyclic adenosine monophosphate response element binding protein/fork- head/winged helix transcription factor 193 （ CREB/Foxp3 ） pathway. Methods Ultracentrifugation was used to isolate exosomes of hepatoeellular carcinoma cell line HepG2 and normal hepatoeellular eel1 line L02. Transmission electron microscopy was used to determine the morphology of exosomes and Western blotting was used to detect the protein markers of exosomes and CREB/Foxp3 expression. HepG2 - exo and L02 - exo were co - cultured with peripheral blood mononuelear cells. The percent of Treg cells were deter- mined by flow cytometry. Results Exosomes was about 30 - 100 nm with a circular or oval shape vesicu- lar structure. CD81 and CD63 were highly expressed in exosomal proteins, while not expressed in cell pro- teins derived from HepG2 or L02 cells. Compared to L02 - exo [ （ 1.87 ± 0. 18 ） % ] and blank control group [ （1.43 ± 0. 12 ）% ], HepG2 -exo strongly increased the percent of Treg ceils [ （6. 87 ± 0. 87 ） % ] , with P value of 0. 005 and 0. 004 respectively. Phosphorylation of CREB in HepG2 - exo group was 1.74 and 1.63 fold higher than it in LO2 - exo and blank control group, and both of the P = 0. 001.Phosphorylation of Foxp3 in HepG2 - exo group was 1.93 and 1.78 fold higher than it in L02 - exo and blank control group, and both of the P = 0. 001. Conclusion These results provided evidence that hepato- cellular carcinoma cell HepG2 derived exosomes could regulates Treg cells via CREB/Foxp3 pathway.

关 键 词：外泌体 调节性T细胞 环磷酸腺苷反应元件结合蛋白 叉状头/翅膀状螺旋 转录因子3 肝癌 

分 类 号：R735.7[医药卫生—肿瘤]