摘要
目的检测黑色素瘤细胞通过外泌体途径上调CD4^+CD25^+调节性T细胞(regulatory T cell,Treg)CXCR3(chemokine receptor 3)受体表达,提高Treg向肿瘤免疫抑制微环境趋化的能力。方法以超速离心法纯化小鼠黑色素瘤细胞B16上清中的外泌体,透射电镜检测外泌体形态,Western blot检测外泌体标记性蛋白的表达。B16细胞荷瘤小鼠。流式细胞术检测荷瘤小鼠(1、2、3周)和尾静脉注射外泌体后脾脏、肿瘤内Treg的频率。磁珠法分离纯化小鼠的Treg;Transwell实验检测Treg的趋化能力。Western blot检测Treg的CXCR3、p-Smad2的表达水平。结果电镜结果显示,B16细胞释放的外泌体为直径30~100 nm的圆形或类圆形结构;高表达标志性蛋白CD63和Alix,不表达Calnexin。在外泌体刺激下Treg的趋化能力显著增强,但可被抗CXCR3的单克隆抗体阻断。Western blot结果显示外泌体可提高Treg细胞内Smad2的磷酸化,并上调CXCR3的表达水平,Treg的趋化能力随之增强。结论B16细胞来源的外泌体通过Smad2磷酸化途径提高了Treg细胞内CXCR3的表达,进而增强其趋化能力。
This study was designed to investigate the effects of melanoma cells on the expression of CXCR3 in CD4^+CD25^+regulatory T cells(Treg)via exosome pathway,and on the chemotaxis of Treg cells to tumor immunosuppressive microenvironment.The exosomes from B16 cells were purified by ultracentrifugation;the morphology of exosomes was detected by transmission electron microscopy(TEM)and the levels of specific proteins were detected by Western blotting.The B16 cell line was used to establish the model of tumor-bearing mice.Flow cytometry was used to detect the frequency of Treg cells in spleen and tumor of the tumor-bearing mice before and after tail vein injection of exosomes.Treg cells were isolated and purified by magnetic beads method,and the chemotaxis of Treg cells were detected by Transwell assay;the protein expression levels of CXCR3 and p-Smad2 in Tregs cells were detected by Western blotting.Data showed that the exosomes released by B16 cells were round or quasi circular in diameter from 30 to 100 nm,and highly expressed CD63 and Alix,but not Calnexin.Exosomes could enhance the chemotaxis of Treg cells significantly,and this effect could be blocked by monoclonal antibodies against CXCR3.Western blot showed that exosomes could significantly enhance the phosphorylation of Smad2 and the expression of CXCR3 in Treg cells.Taken together,exosomes derived from B16 cells can up-regulate the expression of CXCR3 in Treg cells through Smad2 phosphorylationpathway,thusenhancetheirchemotaxis.
作者
颜丽雅
陈欣
薛雅方
YAN Liya;CHEN Xin;XUE Yafang(Department of Dermatology,Taizhou Hospital of Integrated Traditional Chinese and Western Medicine,Taizhou 317523,China;Department of Urology,Taizhou Hospital of Integrated Traditional Chinese and Western Medicine,Taizhou 317523,China;Department of Ultrasound,Southwestern Hospital,Army Medical University,Chongqing 400038,China)
出处
《免疫学杂志》
CAS
CSCD
北大核心
2020年第9期790-795,共6页
Immunological Journal
