【作者】 李建平；

【导师】 韩忠朝；

【作者基本信息】 中国协和医科大学 ， 内科学， 2010， 博士

【摘要】 背景：再生障碍性贫血(aplastic anemia, AA)是一种免疫介导的骨髓衰竭综合征,是血液科常见病、多发病及难治性疾病,其显著特点是骨髓造血衰竭。以往研究更多集中于造血干／祖细胞损伤机制方面,但因其数量显著减少、功能缺陷、体外不易扩增却容易分化等缺点限制此方面研究,目前处于相对停滞状态。寻找一种全新的研究方向势在必行。而再生障碍性贫血患者骨髓脂肪化、血管新生能力降低、骨皮质变薄等现象提示骨髓造血微环境同样存在缺陷。因此,研究骨髓造血微环境中各种细胞成分的前体细胞——骨髓间充质干细胞(bone marrow mesenchymal stem cells, BM-MSCs)的生物学特性及其功能具有重要意义。目的：本研究旨在系统、全面地比较研究再生障碍性贫血患者BM-MSCs和正常人BM-MSCs生物学特性及功能的差异,为探讨再生障碍性贫血BM-MSCs损伤机制提供实验基础及理论依据,亦为再生障碍性贫血的发病机制研究探讨新思路。方法：(1)采用贴壁、传代培养的方法从AA患者和正常人骨髓组织中获取相对纯化的骨髓间充质干细胞；(2)倒置显微镜及共聚焦荧光显微镜观察骨架蛋白β-tubulin染色后BM-MSCs的形态变化；(3)流式细胞仪检测培养的BM-MSCs表面标志；(4)定向诱导BM-MSCs向脂肪细胞、内皮细胞和成骨细胞分化,进行相应染色及分化标志鉴定,比较成脂、成骨、成内皮的分化潜能；(5)测定BM-MSCs成纤维细胞样的集落形成能力(CFU-F)及增殖曲线；(6)流式细胞仪方法检测BM-MSCs细胞周期及细胞凋亡；(7)采用Affymetrix基因表达谱检测方法比较分析AA患者与正常BM-MSCs基因表达谱间的差异；(8)磁珠分选脐血来源的CD34＋细胞,建立BM-MSCs与CD34＋细胞共培养的二种培养体系(LTC assay和MK assay),比较研究AA患者和正常BM-MSCs扩增CD34＋细胞能力和促进造血克隆(CFU-G、CFU-M、CFU-GM、CFU-Mix、BFU-E和CFU-MK)形成的能力；同时比较了二种BM-MSCs表达造血生长因子(SCF、IL-3、EPO、TPO、IL-11、G-CSF、M-CSF和GM-CSF)的异同。(9)磁珠分选健康人外周血CD4＋细胞,同时采用贴壁法去除CD4-细胞中的贴壁细胞以获得相对高比例的CD4-淋巴细胞(大多数为CD8＋细胞),建立BM-MSCs与CD4+／CD4-细胞共培养体系,比较研究BM-MSCs对CD4+／CD4-细胞的克隆形成能力、增殖、分泌TNF-α和IFN-7的影响,以及BM-MSCs促进CD4＋细胞向Treg细胞分化能力。结果：(1)成功培养并获取高纯度的再生障碍性贫血患者骨髓间充质干细胞；(2)AA患者BM-MSCs与正常BM-MSCs存在形态学差异,正常BM-MSCs细胞体积小纤细、呈长梭形,规则排列生长；AA患者BM-MSCs细胞体积较大,呈短梭形、多角形、不规则形,边缘不整齐,部分细胞见双核、多核。(3)AA患者BM-MSCs除了更高表达CD90外,其余表达标志CD73、CD105、CD90、CD44、CD29、CD49e、CD166以及MHC-Ⅰ类分子HLA-ABC无显著差别。二者均不表达造血细胞标志CD34、CD45及MHC-Ⅱ类分子HLA-DR。(4)AA患者BM-MSCs更易向脂肪细胞分化,但分化为内皮细胞和成骨细胞能力弱于正常BM-MSCs。(5)AA患者BM-MSCs形成CFU-F的能力和增殖能力明显弱于正常BM-MSCs。(6)AA患者和正常人BM-MSCs 90%以上均处于G0／G1期,但AA患者BM-MSCs细胞凋亡水平明显增加。(7)Affymetrix基因表达谱检测结果显示,AA患者和正常人BM-MSCs间存在差异表达的基因多达314个(Fold change≥2倍),涉及造血调控、免疫调节、细胞周期、细胞凋亡、细胞粘附、成脂分化等多方面基因。(8)与脐血来源的CD34＋细胞共培养结果显示,AA患者BM-MSCs扩增CD34+细胞能力和促进造血克隆形成能力(尤其支持长期造血能力和巨核细胞系造血能力)减弱。另外,二种BM-MSCs均不表达IL-3和EPO,AA患者BM-MSCs表达SCF、TPO、IL-11 mRNA水平低于正常者,而表达G-CSF和GM-CSF mRNA水平高于正常者,M-CSF差异不显著。(9)与外周血来源的CD4+／CD4-细胞共培养结果显示,AA患者BM-MSCs抑制CD4+／CD4细胞克隆形成、增殖和分泌TNF-a／IFN-γ的能力存在缺陷、促进CD4+细胞向Treg细胞分化能力减弱。结论：(1)再生障碍性贫血患者骨髓间充质干细胞存在多方面、多层次的缺陷。(2)再生障碍性贫血患者BM-MSCs的多种生物学特性异常,包括形态异常、增殖和CFU-F形成能力减弱、多向分化能力异常(易成脂,不易向成骨细胞和内皮细胞方向分化)、细胞凋亡增加、基因表达谱异常。(3)再生障碍性贫血患者BM-MSCs通过分化为成骨细胞和内皮细胞构成HSC龛的能力减弱。(4)再生障碍性贫血患者BM-MSCs支持造血能力(尤其支持长期造血能力和巨核细胞系造血能力)减弱。(5)再生障碍性贫血患者BM-MSCs抑制CD4+／CD4-细胞增殖和分泌TNF-a／IFN-γ能力缺陷、促进CD4＋细胞向Treg细胞分化能力减弱。背景：再生障碍性贫血是一种免疫介导的骨髓衰竭性疾病,其发病机制涉及大量异常的免疫细胞和免疫分子。其中,IFN-γ,、TNF-α和多种白细胞介素(ILs)发挥着主要作用。IL-27是近年发现的一种IL-6／IL-12细胞因子家族成员,在机体内发挥着促进和抑制炎症的双重作用。IL-27可以诱导Th1细胞极化,促进淋巴细胞产生IFN-γ。然而,目前尚无IL-27在再生障碍性贫血发病机制中的相关报道。目的：本研究旨在检测再生障碍性贫血患者骨髓中IL-27及其受体的表达水平,探讨其在再生障碍性贫血发病机制中的作用。方法：(1)采用Ficoll密度梯度离心法分离骨髓单个核细胞(BMMNC):(2)Real-timePCR方法检测BMMNC中IL-27(p28和EBI3)及其受体(WSX-1／TCCR和gp130)mRNA表达水平；(3)流式细胞术检测IL-27刺激前后骨髓CD4+和CD8＋T淋巴细胞内IFN-γ、TNF-a的表达水平；(4)ELISA方法检测骨髓上清中IL-27、IFN-γ和TNF-a的水平以及IL-27刺激前后BMMNC培养上清中IFN-γ和TNF-a的水平变化。结果：(1)再生障碍性贫血患者骨髓BMMNC中IL-27(p28和EB13)和IL-27R的亚单位gp130 mRNA表达水平高于正常对照组(P<0.05),WSX-1／TCCR的表达水平虽然高于正常对照,但无统计学意义(P)>0.05)。(2)再生障碍性贫血患者骨髓上清中IL-27的浓度均高于正常对照(P<0.05),且与疾病的严重程度密切相关。(3)再生障碍性贫血患者骨髓上清中IFN-γ和TNF-a的浓度和CD4＋和CD8＋T淋巴细胞内IFN-γ和TNF-α的表达水平均高于正常对照,除了CD8＋T淋巴细胞内IFN--γ的表达水平无统计学意义外,其余均具有显著性差异(P<0.05)。(4)rhIL-27促进正常对照和再生障碍性贫血患者骨髓CD4＋和CD8＋T淋巴细胞产生更多的TNF-a和IFN-γ(P<0.05)。(5)rhIL-27促进再生障碍性贫血患者BMMNC产生更多的TNF-a和IFN-γ(P<0.05)。结论：再生障碍性贫血患者骨髓中增高的IL-27可能通过促进TNF-a和IFN-γ产生参与其发病机制。更多还原

【Abstract】 Background:Aplastic anaemia (AA) is mostly considered as an immune-mediated bone marrow failure syndrome, characterized by hypoplasia and pancytopenia with fatty bone marrow and reduced angiogenesis. Previous studies have demonstrated that hematopoietic stem cells (HSCs) from AA patients are defective in multiple biological properties and functions. However, too little is known about bone marrow mesenchymal stem cells (BM-MSCs) from patients with aplastic anemia.Objective:This study aims to assess systematically and thoroughly the biological properties and functions of BM-MSCs from patients with aplastic anemia.Methods:(1) BM-MSCs were isolated and purified from bone marrow by adherent culture and passaging in the culture medium (DMEM/DF12 and 2% fetal bovine serum) in vitro. (2) The morphology of BM-MSCs was stained with (3-tubulin and observed by confocal microscopy. (3) The immunophenotype markers of BM-MSCs were detected using flow cytometry. (4) BM-MSCs were induced to differentiate into adipocytes, osteoblasts and endothelial cells, which were identified with corresponding staining and detection of specific genes. (5) The capacity of forming CFU-F and proliferation of BM-MSCs were detected. (6) The cell cycle and apoptosis of BM-MSCs were detected using flow cytometry. (7) The gene profile of BM-MSCs was identified and compared using Affymetrix method. (8) CD34+ cells were purified from umbilical cord by miniMACS immunomagenetic method and co-cultured with BM-MSCs to assess the hematopoiesis-supportive function of BM-MSCs in the amplification of hematopoietic cells and clonogenic potential of CFUs (CFU-G, CFU-M, CFU-GM, CFU-Mix, BFU-E and CFU-MK). The expression of hematopoietic growth factors such as SCF, IL-3, EPO, TPO, IL-11, G-CSF, M-CSF and GM-CSF mRNA were detected by reverse transcriptase-polymerase chain reaction (RT-PCR) method. (9) CD4+ cells were purified from peripheral blood by miniMACS immunomagenetic method and CD4- cells were further purified by adherent culture to remove monocytes, macrophages and dentritic cells. CD4+ cells and CD4- cells were then co-cultured with BM-MSCs to assess the suppression capacity of BM-MSCs in the clonogenic potential and proliferation of lymphocytes and the production of TNF-a and IFN-y by lymphocytes. CD4+ cells were also co-cultured with BM-MSCs to assess the contribution of BM-MSCs in promoting the proliferation of Tregs.Results:(1) We successively isolated and purified BM-MSCs from patients with aplastic anemia. (2) We showed abnormal morphology with irregular appearance using P-tubulin staining, reduced proliferation and clonogenic potential and increased apoptosis of BM-MSCs from AA patients. (3) We demonstrated that BM-MSCs from AA patients were susceptible to be induced to adipocytes but more difficult to osteoblasts and endothelial cells than healthy controls. (4) We also compared the gene expression profile in BM-MSCs from AA patients and healthy controls. A large number of genes associated with hematopoiesis, immune regulation, adipogenesis, apoptosis and cell cycle showed markedly differences between AA patients and healthy controls. (5) Furthermore, we showed that both BM-MSCs from AA patients and healthy controls had the potential of supporting hematopoiesis. But, BM-MSCs from AA patients had reduced potential to amplify CD34+ cells, maintain long-term hematopoiesis. It is more prominent that MSCs from AA patients were markedly defective in supporting the development of megakaryocytes. In addition, we showed that BM-MSCs from AA patients expressed lower levels SCF, TPO and IL-11 mRNA, but higher levels G-CSF and GM-CSF than healthy controls. (6) Finally, we found BM-MSCs from AA were defective in the suppression of proliferation of and clonogenic potential of T cells, TNF-αand IFN-γproduction by CD4+ and CD4- cells.Conclusion:(1) BM-MSCs from AA patients were multiply defective both in biological properties and functions, which might aggravate the bone marrow failure. (2) BM-MSCs from AA were abnormal in morphology and gene profile, defective in clonogenic potential, proliferation and differentiation with increased apoptosis. (3) BM-MSCs from AA were defective in the differentiation into endothelial cells and osteoblasts, which might be the precursor cells in modelling HSC niche. (4) BM-MSCs from AA were defective in maintaining hematopoiesis and supporting the development of megakaryocytes. (5) BM-MSCs from AA were defective in maintaining immune homeostasis. Background:Aplastic anaemia (AA) is considered as an immune-mediated bone marrow failure syndrome. The mechanism is involved with a variety of immune molecules including IFN-y, TNF-a and interleukins (ILs). IL-27 is a novel member of IL-12 family which mediates T cell response and enhances the production of IFN-y. However, little is known about the role of IL-27 in the development of AA.Objective:The present study was aimed to detect the expression of IL-27/IL-27R in bone marrow of patients with aplastic anemia and evaluate the role of IL-27/IL-27R in the pathogenesis of AA.Methods:(1) Bone marrow mononuclear cells (BMMNCs) were isolated from bone marrow using Ficoll-Hypaque gradients centrifugation and cultured in the culture medium (RPMI-1640 and 2% fetal bovine serum) in vitro. (2) The mRNA expression of IL-27 (p28 and EBI3) and IL-27 receptor components (WSX-1/TCCR and gp130) in BMMNCs were detected by real-time reverse transcription polymerase chain reaction (Real-time RT-PCR). (3) The intracellular levels of IFN-y and TNF-a in bone marrow T lymphocytes of patients with AA and healthy controls were measured by flow cytometry. (4) The concentrations of IFN-y and TNF-a in the bone marrow plasma and supernatants of BMMNCs from patients with AA were measured by ELISA.Results:(1) We demonstrated that both the mRNA expression of IL-27/IL-27R subunits in the bone marrow mononuclear cells (BMMNCs) and the levels of IL-27 in the marrow plasma in AA were higher than that observed in controls. (2) Increased IL-27 correlated with the disease severity of AA. (3) Subsequently, we stimulated marrow T lymphocytes with rhIL-27 and found that IL-27 dramatically enhanced the production of TNF-a and IFN-y in both CD4+ and CD8+ T lymphocytes from AA patients. (4) We also detected increased TNF-a and IFN-y in the supernatants of BMMNCs from AA patients after IL-27 stimulation.Conclusion:In conclusion, our data suggest that IL-27 might play an important role in the pathogenesis of AA through enhancing the overproduction of IFN-y and TNF-a.更多还原