靶向成纤维细胞活化蛋白通过影响肿瘤相关成纤维细胞的外泌体抑制内皮细胞间质转化  

Targeting fibroblast activation protein inhibits endothelial-mesenchymal transition by affecting cancer-associated fibroblasts derived exosomes

作　　者：张凯佳 曹望凯 张秀荣[1] 赵厚鑫 崔镓钰 王淑淑 张宝刚 史立宏 ZHANG Kai-jia;CAO Wang-kai;ZHANG Xiu-rong;ZHAO Hou-xin;CUI Jia-yu;WANG Shu-shu;ZHANG Bao-gang;SHI Li-hong(Shandong Province Key Laboratory of Applied Pharmacology,Weifang Medical University,Weifang Shandong 261053,China;Weifang Chest Hospital,Weifang Shandong 261041,China;College of Clinical Medicine,Weifang Medical University,Weifang Shandong 261053,China;College of Rehabilitation Medicine,Weifang Medical University,Weifang Shandong 261053,China)

机构地区：[1]潍坊医学院山东省应用药理学重点实验室,山东潍坊261053 [2]潍坊市胸科医院,山东潍坊261041 [3]潍坊医学院临床医学院,山东潍坊261053 [4]潍坊医学院康复医学院,山东潍坊261053

出　　处：《中国药理学通报》2023年第9期1682-1689,共8页Chinese Pharmacological Bulletin

基　　金：国家自然科学基金面上项目(No 81872163,81672631);潍坊市科技发展计划项目(No 2021YX045)。

摘　　要：目的探讨特异性抑制成纤维细胞活化蛋白(fibroblast activation protein,FAP)是否能够通过影响肿瘤相关成纤维细胞(cancer-associated fibroblasts,CAFs)的外泌体(exosomes,exo)抑制内皮细胞间质转化(endothelial-to-mesenchymal transition,EndMT)并探究其机制。方法提取原代CAFs和癌旁成纤维细胞(peri-tumor fibroblasts,PTFs),收集CAFs-exo和PTFs-exo,特异性FAP抑制剂(3.3 nmol·L^(-1)SP13786)处理CAFs 24 h后收集的外泌体命名为Anti-FAP-exo。将内皮细胞HMEC-1分别以等体积的RPMI 1640、PTFs-exo、CAFs-exo及Anti-FAP-exo孵育并命名为control组、PTF组、CAF组及Anti-FAP组。划痕实验、Transwell侵袭实验、血管生成实验检测各组HMEC-1细胞的迁移、侵袭及血管生成能力;免疫荧光、免疫组化和Western blot检测EndMT相关蛋白表达水平。结果CAF组HMEC-1细胞的迁移、侵袭及血管生成能力较PTF组明显增强,Anti-FAP组HMEC-1细胞迁移、侵袭及血管生成能力较CAF组明显减弱,与PTF组比较无差异。与PTF组相比,CAF组HMEC-1细胞高表达α-SMA、SM22α、p-Stat3和Snail,低表达CD31和VE-cadherin;与CAF组相比,Anti-FAP组HMEC-1低表达α-SMA、SM22α、p-Stat3和Snail,高表达CD31和VE-cadherin。结论特异性抑制FAP会通过影响CAFs外泌体间接抑制血管内皮细胞的迁移侵袭与成管能力,Stat3-Snail-EndMT可能是其潜在机制。Aim To investigate whether targeted inhibition of fibroblast activation protein(FAP)can inhibit the endothelial-to-mesenchymal transition(EndMT)of vascular endothelial cells by affecting exosomes(Exo)of cancer-associated fibroblasts(CAFs)and explore the underlying mechanisms.Methods Primary CAFs and peri-tumor fibroblasts(PTFs)were obtained from lung cancer and peri-cancer tissues,and CAFs-exo and PTFs-exo were collected from culture medium,respectively.Exosomes from CAFs treated with specific FAP inhibitor(3.3 nmol·L^(-1)SP13786)for 24 h were named as Anti-FAP-exo.HMEC-1 cells were incubated in equal volumes of RPMI 1640,PTFs-exo,CAFs-exo and anti-FAP-exo respectively and named as control group,PTF group,CAF group and anti-FAP group.The scratch assay,Transwell invasion assay and angiogenesis assay were used to detect the migration ability,invasion ability and angiogenesis ability of HMEC-1 cells.Immunofluorescence,immunohistochemistry and Western blot were used to detect EndMT-associated protein expression.Results The migration ability,invasion ability and angiogenesis ability of HMEC-1 cells of CAF group were significantly higher than those of PTF group,whereas there was no significant difference between that of anti-FAP group and PTF group.HMEC-1 cells of CAF group had higher expression ofα-SMA,SM22α,p-Stat3 and Snail,and lower expression of CD31 and VE-cadherin than that of PTF group.In addition,HMEC-1 cells of Anti-FAP group had lower expression ofα-SMA,SM22α,p-Stat3 and Snail,and higher expression of CD31 and VE-cadherin than that of CAF group.Conclusions Specific inhibition of FAP could indirectly inhibit the migration ability,invasion ability and angiogenesis ability of vascular endothelial cells via affecting CAFs-exo and Stat3-snail-EndMT pathway may be the potential mechanism.

关 键 词：肿瘤相关成纤维细胞 成纤维细胞活化蛋白 外泌体 内皮细胞 内皮间质转化 血管生成 STAT3 

分 类 号：R329.24[医药卫生—人体解剖和组织胚胎学] R364.3[医药卫生—基础医学] R730.2