他克莫司在治疗重型再生障碍性贫血中的免疫调节作用  

Immune regulatory effect of tacrolimus in patients with severe aplastic anemia and in murine model

作　　者：任悦[1] 李杨[1] 刘春燕[1] 路丹 付蓉[1] Ren Yue;Li Yang;Liu Chunyan;Lu Dan;Fu Rong(Department of Hematology,General Hospital of Tianjin Medical University,Tianjin 300052,China)

机构地区：[1]天津医科大学总医院血液科,天津300052

出　　处：《中华医学杂志》2021年第24期1929-1934,共6页National Medical Journal of China

基　　金：国家自然科学基金(8187011003)。

摘　　要：目的探讨他克莫司在治疗重型再生障碍性贫血(SAA)中的免疫调节作用。方法选取2015年6月至2018年1月天津医科大学总医院血液科确诊的初治SAA患者,应用免疫磁珠分选SAA患者外周血CD8^(+)T细胞,MTT法检测其增殖能力。应用全身照射(TBI)及供者淋巴细胞输注(DLI)方法制备SAA小鼠模型,其中未接受预处理的正常对照组10只;仅接受TBI组10只;接受TBI及DLI组15只。应用流式细胞术检测CD8^(+)T细胞内穿孔素、颗粒酶的表达及SAA小鼠模型中外周循环CD4^(+)/CD8^(+)细胞比例。应用酶联免疫吸附实验(ELISA)检测体外培养CD8^(+)T细胞培养基上清中干扰素γ(IFN‑γ)水平。构建SAA小鼠模型研究各组用药后血象恢复情况及生存时间。结果共纳入初治SAA患者16例,其中男10例、女6例,年龄22~49岁(中位年龄35岁)。在IL‑2浓度20.0、200.0和2000.0 U/ml刺激时他克莫司可抑制CD8^(+)T细胞增殖(P<0.05)。应用他克莫司组SAA患者CD8^(+)T细胞内穿孔素表达低于空白对照组和IL‑2组[(2.25±0.76)%、(6.70±0.82)%比(9.10±1.90)%,均P<0.05]。应用他克莫司组CD8^(+)T细胞IFN‑γ水平低于空白对照组(P<0.05)。给SAA小鼠用药10 d后,他克莫司组SAA小鼠外周血血红蛋白(Hb)、白细胞(WBC)、血小板(PLT)计数均高于SAA组(均P<0.05)。他克莫司组SAA小鼠CD8^(+)T细胞内穿孔素表达低于SAA组[(18.39±6.65)比(29.99±9.83),P<0.05]。SAA组小鼠中位生存时间为18.6 d,90 d存活率为0;他克莫司组SAA小鼠中位生存时间为44.6 d,90 d存活率为80%。他克莫司组SAA小鼠生存时间长于SAA组(P<0.05)。结论他克莫司对SAA患者免疫状态的调节与环孢素A类似,对CD8^(+)T细胞有免疫抑制作用。Objective To investigate the immunomodulatory effect of tacrolimus in severe aplastic anemia(SAA).Methods Patients diagnosed with SAA at the Department of Hematology,General Hospital of Tianjin Medical University from June 2015 to January 2018 were enrolled.CD8^(+)T cells were sorted by immunomagnetic separation from peripheral blood of SAA patients.MTT method was used to detect the proliferation of CD8^(+)T cells.The SAA mouse model was established by total body irradiation(TBI)and donor lymphocyte infusion(DLI).There were 10 normal controls without pretreatment,10 rats in TBI group,15 rats received TBI and DLI.The expression of perforin and granzyme in CD8^(+)T cells and the ratio of CD4^(+)/CD8^(+)cells in peripheral circulation were measured by flow cytometry.The level of interferon-γ(IFN-γ)in medium supernatant of cultured CD8^(+)T cells was tested with enzyme-linked immunosorbent assay(ELISA).SAA mouse model was established to study the recovery of hemogram and survival time after treatment.Results A total of 16 SAA patients were enrolled,and there were 10 males and 6 females,with a median age of 35(22-49)years.Tacrolimus inhibited the proliferation of CD8^(+)T cells when IL-2 concentration was 20.0,200.0 and 2000.0 U/ml(P<0.05).The expression of perforin in CD8^(+)T cells of SAA patients treated with tacrolimus was significantly lower than that in blank control group and IL-2 group[(2.25±0.76)%,(6.70±0.82)%vs(9.10±1.90)%,all P<0.05].The level of IFN-γin CD8^(+)T cells group after applying tacrolimus was significantly lower than that in the blank control group(P<0.05).After 10 days of administration,the peripheral blood hemoglobin(Hb),white blood cell(WBC)and platelet(PLT)counts of SAA mice in tacrolimus group were all higher than those in SAA group(all P<0.05).The expression of perforin in CD8^(+)T cells in tacrolimus group was significantly lower than that in SAA group[(18.39±6.65)vs(29.99±9.83),P<0.05].The median survival time of SAA group was 18.6 days,and the 90 day survival rate was 0.The medi

关 键 词：贫血 再生障碍性 他克莫司 CD8^(+)T淋巴细胞 

分 类 号：R556.5[医药卫生—血液循环系统疾病]