体外光化学疗法预防小鼠急性移植物抗宿主病作用及其机制  

Effect and mechanism of extracorporeal photopheresis on preventing acute graft versus host disease in ice

作　　者：程占睿 林雨薇 钟妍 张阿龙 徐海霞 田力 刘忠[1,2] CHENG Zhanrui;LIN Yuwei;ZHONG Yan;ZHANG Along;XU Haixia;TIAN Li;LIU Zhong(Institute of Blood Transfusion,Chinese Academy of Medical Sciences&Peking Union Medical College,Chengdu 610052,China;Key Laboratory of Transfusion Adverse Reactions,Chinese Academy of Medical Sciences;School of Public Health,Anhui Medical University.)

机构地区：[1]中国医学科学院北京协和医学院输血研究所,四川成都610052 [2]中国医学科学院输血不良反应研究实验室 [3]安徽医科大学公共卫生学院

出　　处：《中国输血杂志》2023年第10期860-867,共8页Chinese Journal of Blood Transfusion

基　　金：血液安全保障及新产品新技术研究,中国医学科学院医学与健康科技创新工程(2021-1-I2M-060);输血不良反应发生机制与干预,四川省国际科技创新合作/港澳台科技创新合作项目(2020YFH0024)。

摘　　要：目的探讨体外光化学疗法(extracorporeal photopheresis,ECP)诱导的早期凋亡脾脏单个核细胞对小鼠急性移植物抗宿主病(acute graft versus host disease,aGVHD)的预防效果并初步探究其作用机制。方法1)提取C57BL/6小鼠的脾脏单个核细胞,用不同浓度8-MOP(50 ng/mL,100 ng/mL,200 ng/mL,300 ng/mL,600 ng/mL)处理后2J/cm2的紫外线照射,通过Annexin V-FITC/PI凋亡检测试剂盒检测细胞的早期凋亡率,确定实验最适宜的8-MOP浓度。2)取6~8周龄SPF级雌性BALB/C小鼠(H-2Kd)35只,X射线8Gy全身照射后分为5组:单纯照射组尾静脉回输生理盐水0.2 mL;同基因骨髓移植组尾静脉回输BALB/C小鼠骨髓有核细胞悬液0.2 mL(含细胞量1×10^(7));异基因骨髓移植组尾静脉回输C57BL/6小鼠骨髓有核细胞悬液0.2 mL(含细胞量1×10^(7));aGVHD组尾静脉注射C57BL/6小鼠骨髓有核细胞(含细胞量1×10^(7))和脾脏单个核细胞(含细胞量1×10^(7))混合液0.2 mL;ECP预防aGVHD组移植前48h预先尾静脉回输ECP处理过的C57BL/6小鼠脾脏单个核细胞0.2 mL(含细胞量1×10^(7)),移植当天尾静脉注射C57BL/6小鼠骨髓有核细胞(含细胞量1×10^(7))和脾脏单个核细胞(含细胞量1×10^(7))混合液0.2 mL。观察ECP对aGVHD的预防效果,应用CBA法检测各组小鼠血清IFN-γ,IL-2、TNF、IL-4和IL-6浓度;流式细胞术检测Th1类细胞的计数。结果不同浓度8-MOP(50 ng/mL,100 ng/mL,200 ng/mL,300 ng/mL,600 ng/mL)处理后小鼠脾脏单个核细胞的早期凋亡率(%):(14.18±0.865)vs(16.76±0.407)vs(18.83±0.404)vs(19.27±0.404)vs(14.5±0.529),确定8-MOP的最适宜浓度为200 ng/mL。在体内实验中,aGVHD组较同基因骨髓移植组和异基因骨髓移植组生存率下降、体重减低、临床评分增高(P<0.01,P<0.01),且移植后小鼠骨髓细胞嵌合率大于90%;ECP可以显著改善移植后小鼠存活率,降低临床评分(P<0.05),并降低血清Th1类细胞因子浓度(P<0.05)和脾脏中Th1类细胞的计数(P<0.05)。结论ECP诱导的�Objective To investigate the preventive effects of early apoptotic splenic mononuclear cells induced by extracorporeal photopheresis(ECP)on acute graft versus host disease(aGVHD)in mice and explore the underlying mechanisms.Methods 1)Splenic mononuclear cells were extracted from C57BL/6 mice and treated with different concentrations of 8-MOP(50 ng/mL,100 ng/mL,200 ng/mL,300 ng/mL,600 ng/mL).After treatment,irradiate the cells with 2 J/cm2 of ultraviolet light.Then,use the Annexin V-FITC/PI apoptosis detection kit to assess the early apoptosis rate of the cells and determine the optimal concentration of 8-MOP for the experiment.2)There were 35 SPF-grade female BALB/C mice(H-2Kd)aged 6-8 weeks.After whole-body irradiation with 8Gy X-rays,the mice were divided into five groups:sham irradiation group received intravenous infusion of 0.2 mL of normal saline,the syngeneic bone marrow transplantation group received intravenous infusion of 0.2 mL of BALB/C mouse bone marrow nucleated cell suspension(including a cell count of 1×10^(7)),the allogeneic bone marrow transplantation group received intravenous infusion of 0.2 mL of C57BL/6 mouse bone marrow nucleated cell suspension(including a cell count of 1×10^(7)),the aGVHD group received intravenous injection of a mixture of C57BL/6 mouse bone marrow nucleated cells(including a cell count of 1×10^(7))and splenic mononuclear cells(including a cell count of 1×10^(7))in 0.2 mL,the ECP prevention group received pre-transplant intravenous infusion of 0.2 mL of ECP-treated splenic mononuclear cells of C57BL/6 mice(including a cell count of 1×10^(7))48 hours before transplantation,and on the day of transplantation,intravenous injection of a mixture of C57BL/6 mouse bone marrow nucleated cells(including a cell count of 1×10^(7))and splenic mononuclear cells(including a cell count of 1×10^(7))in 0.2 mL.The preventive effects of ECP on aGVHD were observed,and the concentrations of IFN-γ,IL-2,TNF,IL-4 and IL-6 in mouse serum were measured using CBA.Th1 cell counts were determ

关 键 词：体外光化学疗法 异基因造血干细胞移植 急性移植物抗宿主病 细胞疗法 

分 类 号：R457.1[医药卫生—治疗学] R453[医药卫生—临床医学] R318.06