雷帕霉素对JAK2 V617F阳性HEL细胞增殖凋亡及PD-1/PD-L1信号通路的影响  

Effects of Rapamycin on Proliferation Apoptosis and PD-1/PD-L1 Signaling Pathway of JAK2 V617F Positive HEL Cells

作　　者：齐林 王蕊[2] 谢旭磊[3] 张朝 成志勇[3] 付建珠 QI Lin(Chengde Medical University, Hebei Chengde 067000, China)

机构地区：[1]承德医学院研究生院,河北承德067000 [2]河北省石家庄市第四医院内科,河北石家庄050000 [3]河北省保定市第一医院血液内科,河北保定071000

出　　处：《河北医学》2022年第5期705-710,共6页Hebei Medicine

基　　金：河北省重点科技计划项目,(编号:162777120D)。

摘　　要：目的:探究雷帕霉素(Rapa)对JAK2 V617F阳性HEL细胞增殖凋亡及程序性死亡受体1与其配体(PD-1/PD-L1)的影响。方法:体外培养JAK2 V617F突变阳性的人红白血病HEL细胞,分别加入浓度为10nM、50nM和100nM的Rapa,设立对照组,CCK-8检测细胞增殖抑制率。Caspase 3/7活性检测试剂盒检测各组细胞Caspase 3/7活性,Transwell小室观察各组细胞迁移能力,流式细胞术检测各组细胞凋亡情况及PD-1/PD-L1表达情况,实时荧光定量聚合酶链反应检测各组细胞中JAK2和PD-1、PD-L1 mRNA变化,蛋白质印迹法检测各组哺乳类雷帕霉素靶蛋白(mTOR)的表达。选取5名健康志愿者,分离其淋巴细胞与HEL细胞共培养,加入不同浓度的Rapa,流式细胞术检测各组Treg细胞数量变化。结果:CCK-8检测结果显示:不同终浓度Rapa(10nM、50nM、100nM)在72h时对细胞增殖抑制率分别为(33.33±4.6)%、(49.12±3.72)%、(55.16±4.14)%(P<0.05)。在浓度50nM和100nM的Rapa作用HEL细胞24h后,其Caspase 3/7活性较对照组显著升高(均P<0.05),流式细胞术结果显示与对照组相比细胞凋亡率明显增加(P<0.05),PD-L1蛋白表达较对照组明显下降(P<0.05),JAK2和PD-L1 mRNA表达量较对照组显著下调(P<0.01)。健康志愿者淋巴细胞与HEL细胞共培养24h后,其Treg细胞增加(P<0.05),而加入Rapa可使Treg细胞呈剂量依赖性下调(P<0.05)。蛋白质印迹法检测结果显示Rapa能够剂量依赖性抑制mTOR蛋白表达。结论:Rapa可能通过影响mTOR进而干扰JAK2通路,导致HEL细胞增殖受抑,PD-L1表达减低,Treg细胞受抑。Objective:To explore the effect of Rapamycin(Rapa)on proliferation and apoptosis of JAK2 V617F positive HEL cells and the expression of programmed death receptor 1 and its ligand(PD-1/PD-L1).Methods:Human erythroleukemia HEL cells with JAK2 V617F mutation positive were cultured in vitro.Rapa with concentrations of 10nm,50nm and 100nm were added respectively.The control group was established.The inhibition rate of cell proliferation was detected by CCK-8.Caspase 3/7 activity detection kit was used to detect the caspase 3/7 activity of cells in each group,Transwell chamber was used to observe the cell migration ability of each group,flow cytometry was used to detect the apoptosis and PD-1/PD-L1 expression of cells in each group,real-time fluorescence quantitative polymerase chain reaction was used to detect the mRNA changes of JAK2,PD-1 and PD-L1 in cells in each group,and Western blot was used to detect the expression of mammalian rapamycin target protein(mTOR)in each group.Five healthy volunteers were selected.Their lymphocytes were isolated and co cultured with HEL cells.Different concentrations of Rapa were added.The number of Treg cells in each group was detected by flow cytometry.Results:Results of CCK-8 showed that:The inhibition rates of cell proliferation at 72h with different final concentrations of Rapa(10 nM,50 nM,100 nM)were(33.33±4.6)%,(49.12±3.72)%,(55.16±4.14)%(P<0.05),respectively.Rapa treated HEL cells at concentrations of 50nM and 100nM after 24h,Caspase 3/7 activity was significantly increased compared with control(P<0.05).The results of flow cytometry showed that the apoptotic rate increased significantly compared with the control.PD-L1 expression was significantly decreased compared with the control(P<0.05).The mRNA expression of JAK2 and PD-L1 was significantly downregulated compared with the control(P<0.01).Healthy volunteer lymphocytes co-cultured with HEL cells for 24h showed an increase in Treg cells(P<0.05).Rapa could reduce Treg cells in a dose-dependent manner.Western blot showed th

关 键 词：雷帕霉素 JAK2 V617F 程序性死亡受体1 

分 类 号：R73[医药卫生—肿瘤]