CD200/CD200R在异基因造血干细胞移植后的表达水平及意义

【作者】 黄月华；

【导师】 周道斌；

【作者基本信息】 北京协和医学院 ， 内科学（专业学位）， 2014， 博士

【摘要】 背景和目的CD200与其受体CD200R均为跨膜糖蛋白,均属于免疫球蛋白超家族的成员,CD200在体内存在广泛。CD200与CD200R的相互作用对于免疫调节的影响已经在动物以及体外实验中得到了证实,除了对巨噬细胞具有抑制作用以外,有研究表明,在调节性T细胞的调节、过敏反应、自体免疫、同种异体移植、神经系统疾病以及其他一些自身免疫性疾病中,CD200/CD200R均发挥着重要的作用。CD200在急性白血病、多发性骨髓瘤及慢性淋巴细胞白血病等恶性疾病中高表达,且与预后相关,因此可将CD200作为这些疾病的预后因素.CD200/CD200R与异基因造血干细胞移植之间的关系鲜有报道,因此本文主要对CD200及其受体在移植物抗宿主病(graft versus host disease, GVHD)中的作用进行探讨。研究方法实验标本来源于2013年2月至2013年12月在北京协和医院就诊的异基因造血干细胞移植患者。首先应用流式细胞术测定各研究组患者细胞表面CD200及CD200R在CD19+细胞、CD3+细胞及树突状细胞(CD19-CD14-CD1c+)表面表达水平的差异。随后通过应用实时荧光定量聚合酶链反应技术在mRNA水平对各研究组CD200及CD200R的表达量进行检测,观察各组间mRNA表达水平的差异。最后将各研究组外周血单个核细胞与抗-CD200R1抗体体外共培养48h,采用酶联免疫吸附剂测定法测定培养上清的白介素-10水平,间接验证CD200/CD200R的功能。研究结果1、aGVHD组和non-aGVHD组CD19+CD200+细胞的表达水平低于正常对照组。non-aGVHD组CD19+CD200+细胞的表达水平高于aGVHD组(P<0.05)。2、 cGVHD组CD19+CD200+细胞的表达水平与正常对照组无差异,non-cGVHD组CD19+CD200+细胞的表达水平高于正常对照组。non-cGVHD组CD19+CD200+细胞的表达水平高于cGVHD组。(P<0.05)3、CD3+细胞和树突状细胞CD200及CD200R的表达在各研究组间无差异。4、 aGVHD组和cGVHD组CD200的mRNA表达水平均低于正常对照组(P<0.05)。5、 aGVHD组CD200的mRNA表达水平低于non-aGVHD组,cGVHD组低于non-cGVHD组(P<0.05)。6、 non-aGVHD组患者CD200的mRNA的表达水平与正常对照组无差异,non-cGVHD组高于正常对照组(P<0.05)。7、CD200R的mRNA的表达水平在各研究组间未见明显差异。8、抗-CD200R1抗体与各实验组外周血单个核细胞体外共培养48h后,细胞上清IL-10浓度随抗-CD200R1抗体浓度的增加呈下降趋势。结论研究显示,发生GVHD的患者CD200的]mRNA表达水平明显低于同时间点未发生GVHD患者及正常对照组。aGVHD组患者CD19+CD200+细胞表达水平低于non-aGVHD组,cGVHD组低于non-cGVHD组。提示CD200与GVHD的发生可能有一定的相关性,CD19+CD200+细胞在GVHD的致病机制中发挥着一定的作用。更多还原

【Abstract】 Background and ObjectiveCD200and its receptor CD200R are both type-1membrane glycoproteins, which are members of the immunoglobulin superfamily (IgSF). CD200is widely expressed on a variety of cell types, while the recently identified CD200R is expressed on myeloid cells and T cells. The immunomodulatory effects of CD200and CD200R interactions have been demonstrated in animal models and in vitro experiments. Besides the inhibition effects on macrophages, CD200/CD200R also play an important role in regulating the regulatory T cells, allergicreaction, autoimmune diseases, allograft, neurological diseases and other autoimmune-related diseases, etc. CD200is highly expressed in patients with acute leukemia, multiple myeloma, chronic lymphocytic leukemia and other malignant diseases. CD200is considered to be associated with the prognosis of these diseases, and it could be used as an independent prognostic factor. The relationship between CD200/CD200R and allogeneic hematopoietic stem cell transplantation has been reported rarely. Therefore, this paper mainly focuses on the role of CD200and its receptor in the graft versus host disease (GVHD).MethodsExperimental samples were collected from the allogeneic hematopoietic stem cell transplantation patients who were treated in Peking Union Medical College Hospital from February2013to December2013. Firstly, the expression levels of CD200and CD200R on CD19+cell, CD3+cell and dendritic cell (CD19-CD14-CDlc+) surface in each research group were detected by using flow cytometry, to determine whether there were expression differences among each group. Then, the mRNA levels of each research group were tested by using real-time quantitative polymerase chain reaction, to find the differences of mRNA expression levels among each group. Finally, the peripheral blood mononuclear cells of the patients and healthy controls were co-cultured with anti-CD200R1antibody for48hours, and the interleukin-10levels in the co-culture system were tested by using enzyme linked immunosorbent assay, to indirectly verify the function of CD200/CD200R.Results1、 The CD200expression levels on CD19+cells surface in the aGVHD group and non-aGVHD group were both lower than healthy control group.The CD200expression levels on CD19+cells surface in the non-aGVHD group were higher than the aGVHD group(P<0.05).2、There was no significant difference of the expression levels of CD19+CD200+cells between the cGVHD group and healthy control group. The expression levels of CD19+CD200+cells in the non-cGVHD group were higher than the cGVHD group and healthy control group(P<0.05).3、 There were no significant differences of CD200and CD200R expression levels on CD3+cells and dendritic cells among all research groups.4、 The CD200mRNA expression levels in the aGVHD group and cGVHD group were both lower than the healthy control group(P<0.05).5、 The CD200mRNA expression levels were lower in the aGVHD group than the non-aGVHD group, and were lower in the cGVHD group than the non-cGVHD group(P <0.05).6、 The CD200mRNA expression levels had no significant difference between the non-aGVHD patients and the healthy control group, but were higher in the non-cGVHD group than the healthy control group (P<0.05).7、There was no significant difference of the CD200R mRNA expression levels among all research groups.8、 After the peripheral blood mononuclear cells of the patients and healthy controls were co-cultured with anti-CD200R1antibody for48hours, the interleukin-10concentration decreased with the increasing concentrations of anti-CD200R1antibody in the co-culture system.ConclusionThe study showed that the patients with GVHD had a significantly lower CD200mRNA levels than the patients without GVHD and the healthy control group. The CD200expression levels of CD19+cells in the patients with aGVHD were also lower than the non-aGVHD group. These results indicate that CD200and CD19+CD200+cells play a role in the pathogenesis of GVHD.更多还原