造血微环境免疫状态对IRHS骨髓单个核细胞端粒酶活性的影响及临床意义

Effect and clinical significance of immunological state upon telomerase activation of bone marrow mononuclear cells in hemopoietic microenvironment of immune related hematocytopenia(IRHS)

目的:观察免疫相关性血细胞减少综合征(Immune related hematocytopenia syndrome,IRHS)患者骨髓造血微环境免疫分子表达状态,对骨髓单个核细胞(BMMNC)端粒酶活性(TA)的影响,探讨对造血细胞破坏的免疫损伤机制及临床意义。方法:①端粒重复序列扩增-酶联免疫吸附法(TRAP-PCR-ELISA)检测366例IRHS患者治疗前后BMMNC的TA。②免疫化学染色和免疫荧光染色,观察患者骨髓细胞和造血微环境中HLA-DR、抗人IgG、FcγⅡ受体(FcγⅡR)、甘露糖受体(MR)、白细胞介素17A(IL-17A)及受体表达。③FACS技术检测患者外周血淋巴细胞CD3+CD4+T细胞、CD3+CD8+T细胞、CD3-CD16+/CD56+NK细胞和CD19+B细胞比例。60例健康查体者外周血淋巴细胞亚群作为正常对照,30例缺铁性贫血患者骨髓作为疾病对照。结果:①IRHS患者治疗前端粒酶活性为0.2617±0.0216,高于疾病对照组(0.0616±0.0313,P<0.01);其中HLA-B27+组高于HLA-B27-组(0.3013±0.0206比0.1923±0.0129,P<0.05),HLA-B27+IgG升高的重度IRP患者显著高于HLA-B27-IgG升高组(0.4016±0.0172比0.2211±0.0110,P<0.01)。②HLA-B27+IgG+组骨髓免疫细胞和造血微环境呈现细胞免疫活化和体液免疫均紊乱状态,高表达HLA-DR、FcγⅡR、IL-17A、IL-17RA和MR等免疫分子,外周血CD3+CD4+T细胞、CD3+CD8+T细胞和CD3-CD16+/CD56+NK和CD19+B细胞比例呈不同程度升高,造血破坏呈现多样性。③而HLAB27-IgG+组骨髓免疫细胞和造血微环境呈现体液免疫活化优势状态,高表达FcγⅡR,IL-17A等炎性因子不表达或弱表达,外周血仅CD19+B细胞比例升高,骨髓造血破坏方式主要为抗体依赖性细胞毒性(ADCC)效应。经糖皮质激素联合环孢素A等药物治疗后,患者血象恢复时BMMC的端粒酶重新失活,TA为0。结论:IRHS骨髓单个核细胞端粒酶活化,与患者骨髓造血微环境免疫状态和病变血细胞病理性损害程度密切相关。病毒感染和细胞免疫活化,多种炎性因子参与IRHS造血免疫损伤,是TA增强的重要因素。

Objective：To study the effect of immunological molecules expressive state upon the telomerase activation （ TA） of bone marrow mononuclear cells （ BMMNC ） in the hemopoietic microenvironment of patients with immune related hematocytopenia （ IRHS） ,and to explore the immunologic mechanism as well as the clinical significance of hematoclasis in marrow of IRHS patients .Methods：①TRAP-PCR-ELISA method was performed to detect the TA of BMMNC in marrow of 366 IRHS patients before and after therapy.②The molecules HLA-DR,anti-hunman IgG,FcγⅡR,mannose receptor （ MR）,IL-17A and its receptor （ IL-17AR） were analysed by immunochemistry and immunofluorescence staining .③The flow cytometric （ FCM） was used to analyse the proportion of CD3＋CD4^＋T cells as well as CD3＋CD8＋T cells ,CD3-CD19＋B cells and CD3-CD16/56＋NK cells in peripheral blood lymphocyte.60 cases of health examination were selected as the control group , and 30 cases hypoferric anemia patients were selected as disease control.The differences between patient group and control group were analysed with statistic method .Immunochemistry and immunofluorescence staining were performed to in situ analyze the activation-characteristics of immunocyte in bone marrow slides of IRHS ,and the dependablity of cellular immunologic injury was also checked.Results： ①The levels of TA was 0.261 7 ±0.021 6 before treatment , higher than the disease control group （0.061 6±0.031 3 ,P〈0.01）.Among of them HLA-B27＋patients were higher than HLA-B27-patients （0.301 3±0.020 6 vs.0.192 3±0.012 9,P〈0.05）.Serious IRP patients with HLA-B27＋IgG＋were obviously higher than HLA-B27-IgG＋patients （0.401 6±0.017 2 vs.0.221 1±0.011 0,P〈0.01）.②In marrow of HLA-B27＋IgG＋patients,both cell immunity and humoral immunity were in disorder in the hemopoietic microenvironment ,and immonocyte in marrow expressed HLA-DR, FcγⅡR,IL-17A,IL-17RA and MR,and Th,Ts,B cell and NK cell in peripheral blood increased in different degree ,inducing the in-flammation of haemocyte and lead to destruction.③Humoral immunity was in the dominant level in morrow;humoral immunity of HLA-B27-IgG＋patients,immonocyte expressed FcγⅡR in high level,but IL-17A was seldom expressed,only CD19＋B cell was increased slightly ,the antibody dependent cellular cytotoxicity （ ADCC） was the main mode of destruction.After therapy glucocorticoids associated with ciclosporin A ,the TA level of BMMNC decreased to 0 with devitalization.Conclusion： The telomerase activation of bone marrow mononuclear cells in IRHS is related with the immune state of hemopoietic microenvironment and the pathologic lesion degree of hema -topoietic cell in marrow.It is viral infection and immunological activation as well as a variety of inflammatory factors play a part in the immunologic injury that might be an important factor of the enhancement in TA.