摘要
目的探讨Renca细胞来源的外泌体(exosomes)活化的髓源抑制性细胞(myeloid-derived suppressor cells, MDSCs)对肾癌抗原特异性细胞毒性T细胞(cytotoxic lymphocytes, CTLs)体外增殖和杀伤效应的抑制作用。方法超速离心法从Renca细胞无血清上清液中提取出exosomes,流式细胞术检测exosomes对MDSCs的活化效应。分离并培养小鼠树突状细胞(dendritic cells, DCs)和CD8+T细胞,制备DCs活化的肾癌抗原特异性CTLs。磁珠分选exosomes活化的MDSCs,羧基荧光素乙酰乙酸琥珀酰亚胺酯(carboxyfluorescein diacetate succinimidyl ester,CFSE)荧光染色和流式细胞术检测MDSCs对CTLs增殖的抑制作用。将MDSCs、CTLs和Renca细胞体外共培养,乳酸脱氢酶(lactate dehydrogenase, LDH)释放实验检测exosomes活化的MDSCs对CTLs杀伤效应的抑制作用。结果 Renca细胞来源的exosomes有效地诱导了MDSCs的聚集,成功制备了DCs并获得了DCs诱导的肾癌抗原特异性CTLs。与exosomes活化的MDSCs共培养后的CTLs其增殖指数和杀伤率明显低于与未活化的MDSCs共培养后的CTLs和单独的CTLs(P<0.05)。结论 Renca细胞来源的exosomes活化的MDSCs抑制了肾癌抗原特异性CTLs的体外增殖和杀伤效应,可能在肾癌免疫逃逸中扮演重要作用。
Objective To investigate the inhibitory effect of myeloid-derived suppressor cells(MDSCs)activated by exosomes derived from Renca cells on the proliferation and killing activity of renal cancer antigen-specific cytotoxic T lymphocytes(CTLs)in vitro.Methods Exosomes derived from Renca cells were purified by ultracentrifugation,and exosome-induced activation of MDSCs was detected using flow cytometry.Mouse dendritic cells(DCs)and CD8^+T cells were isolated to prepare DC-activated renal cancer antigen-specific CTLs.Magnetic beads were used to sort out MDSCs activated by the exosomes,and fluorescence staining with carboxyfluorescein diacetate succinimidyl ester(CFSE)and flow cytometry were performed to assess the inhibitory effect of activated MDSCs on the proliferation of CTLs.MDSCs,CTLs and Renca cells were co-cultured in vitro,and the inhibitory effect of exosomes-activated MDSCs on the killing activity of CTLs was observed using LDH release assay.Results Renca cell-derived exosomes effectively induced the aggregation of MDSCs.We successfully isolated DCs from the mice and obtained DC-activated renal cancer antigen-specific CTLs.Compared with the CTLs cultured alone or in the presence of unactivated MDSCs,the CTLs co-cultured with exosomes-activated MDSCs showed a significantly lower proliferation index and a lower killing rate against Renca cells(P<0.05).Conclusion MDSCs activated by Renca cell-derived exosomes can inhibit the proliferation and lower the cell-killing efficiency of renal cancer antigen-specific CTLs in vitro,suggesting a significant role of MDSCs in immune escape of renal cancer.
作者
徐浩语
王和西
黎楠
张尧
XU Haoyu;WANG Hexi;LI Nan;ZHANG Yao(Department of Urology,the First Affiliated Hospital of Chongqing Medical University,Chongqing,400016,China)
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2019年第10期923-930,共8页
Journal of Third Military Medical University
基金
国家自然科学基金面上项目(81272572)~~
