雷帕霉素对人红白血病HEL细胞外泌体及PD-1/PD-L1的影响  

Effect of Rapamycin on Exosomes and PD-1/PD-L1 in Human Erythroleukemia HEL Cells

作　　者：齐林 张朝 王素云 刘贵敏[3] 王蕊[2] 付建珠 成志勇[3] QI Lin;ZHANG Zhao;WANG Suyun;LIU Guimin;WANG Rui;FU Jianzhu;CHENG Zhiyong(Graduate School,Chengde Medical University,Chengde 067000,China;Department of Internal Medicine,The 4th Hospital of Shijiazhuang,Shijiazhuang 050000,China;Department of Hematology,Baoding No.1 Hospital,Baoding 071000,China;Department of Hematology,Shenzhen Longhua District Central Hospital,Shenzhen 518000,China)

机构地区：[1]承德医学院研究生院,承德067000 [2]石家庄市第四医院内科,石家庄050000 [3]保定市第一医院血液内科,保定071000 [4]深圳市龙华区中心医院血液内科,深圳518000

出　　处：《肿瘤防治研究》2022年第10期1021-1027,共7页Cancer Research on Prevention and Treatment

基　　金：河北省重点研发计划项目(223777105D)。

摘　　要：目的探讨雷帕霉素(Rapa)对JAK2 V617F阳性HEL细胞外泌体中JAK2、ABCA3及免疫检查点PD-1/PD-L1的影响。方法体外培养人红白血病HEL细胞(JAK2 V617F阳性),分别加入浓度为10、50和100 nmol/L的Rapa,设立对照组。CCK-8检测细胞增殖抑制率,最终确定细胞增殖抑制率选用浓度为10及50 nmol/L的Rapa干预细胞。外泌体试剂盒提取外泌体,Western blot及流式细胞术鉴定外泌体,荧光定量PCR检测外泌体中JAK2、ABCA3和PD-1、PD-L1 mRNA变化,流式细胞术检测外泌体PD-1/PD-L1蛋白表达。结果外泌体试剂盒提取的外泌体均表达特征性的CD9、CD63、CD81蛋白,符合外泌体一般特征。HEL细胞外泌体中能够检测到JAK2 mRNA;Rapa能够减少HEL细胞外泌体产生,剂量依赖性降低外泌体中JAK2、ABCA3及PD-L1相对表达量,抑制外泌体PD-L1蛋白表达,对PD-1蛋白表达无明显影响。结论HEL细胞可能通过外泌体传递JAK2突变基因信号,Rapa能够减少外泌体产生,进而抑制外泌体传递JAK2突变信号,抑制PD-L1蛋白表达。Objective To determine the effect of rapamycin(Rapa)on JAK2,ABCA3,and the immune checkpoint PD-1/PD-L1 in exosomes derived from JAK2 V617F positive HEL cells.Methods Human erythroleukemia HEL cells(JAK2 V617F mutation-positive)were cultured in vitro,and rapamycin was added at concentrations of 10,50,and 100 nmol/L.The control group was established and cell proliferation inhibition rate was detected by CCK-8.Based on the inhibition rate of cell proliferation,cells intervened with 10 and 50 nmol/L Rapa were selected.Exosomes were extracted using a kit and identified by Western blot and flow cytometry.JAK2,ABCA3,and PD-1/PD-L1 mRNA changes in exosomes were detected by fluorescent quantitative PCR.The expression of exosome PD-1/PD-L1 protein was determined by flow cytometry.Results The exosomes extracted with the exosome kit all expressed characteristic CD9,CD63,and CD81 proteins,which were consistent with the general characteristics of exosomes.JAK2 mRNA can be detected in exosomes from HEL cells;Rapa reduced exosome production by HEL cells,and dose-dependently decreased gene expression of JAK2,ABCA3,and PD-L1 in exosomes.In addition,Rapa inhibited exosomal PD-L1 protein expression and had no significant effect on PD-1 protein expression.Conclusion HEL cells may transmit JAK2 mutant gene signals via exosomes.Rapa can reduce the production of exosomes and inhibits JAK2 mutated signals delivered by exosomes and suppresses PD-L1 protein expression.

关 键 词：雷帕霉素 外泌体 HEL细胞 PD-1/PD-L1 

分 类 号：R733.7[医药卫生—肿瘤]