【作者】 罗成燕；

【导师】 李大金； 王凌；

【作者基本信息】 复旦大学 ， 妇产科学， 2009， 博士

【摘要】 骨骼系统和免疫系统共享许多调节分子,包括细胞因子及其受体、信号分子和转录因子。此外,骨细胞和免疫细胞处于共同的微环境骨髓腔中,免疫应答所产生和释放的各种细胞因子显著影响骨细胞功能和骨代谢。在生理和病理过程中,尤其是自身免疫性疾病和其他炎症性疾病中,与破骨细胞紧密关联的骨系统和免疫系统之间的联系更为密切。在2000年Arron和Choi将之命名为“骨免疫学”。破骨细胞在骨重建这个生理过程和一些疾病,如骨质疏松、类风湿性关节炎以及牙周病中发挥着重要的作用。破骨细胞来源于单核巨噬细胞系统的造血干细胞,并在巨噬细胞集落刺激因子（macrophage colony-stimulating factor,M-CSF）和RANKL（receptor activator of nuclear factor kappa B ligand,RANKL）的作用下,分化为多核的成熟破骨细胞（osteoclast,OC）。在风湿性疾病和炎症性肠病观察到激活的T细胞表达RANKL,进而OC形成增加;因此,免疫细胞调节着OC功能及骨吸收。骨质疏松症被认为是一种慢性炎症性疾患。第一部分调节性T细胞效应性细胞因子IL-10、TGF-β1对破骨细胞分化与骨吸收的抑制作用目的调节性T细胞主要通过分泌IL-10与TGF-β1发挥其免疫调节作用;本研究拟探讨调节性T细胞效应性细胞因子IL-10与TGF-β1对人胚骨髓细胞来源的破骨细胞分化与骨吸收功能的调控作用。方法人骨髓细胞取自孕13—16周药物流产胚胎,经M-CSF与RANKL体外诱导分化成OC细胞。靶细胞经不同浓度的IL-10与TGF-β1处理后,用TRAP染色观察破骨细胞分化;用甲苯胺兰染色骨吸收陷窝,以观察其骨吸收作用。结果经浓度为50ng/ml的IL-10或TGF-β1处理后,能明显抑制破骨细胞分化与骨吸收作用;进一步研究发现,IL-10或TGF-β1对OC细胞的调控作用呈明显的剂量依赖性。IL-10与TGF-β1联合处理时,在10ng/ml时就显示出对破骨细胞分化与骨吸收作用的明显抑制,表明IL-10与TGF-β1对破骨细胞分化与骨吸收的抑制作用有显著的协同效应。结论调节性T细胞效应性细胞因子IL-10与TGF-β1对破骨细胞分化与骨吸收功能具有抑制作用;并且两者呈现协同效应。第二部分CD4⁺CD25⁺Foxp3⁺调节性T细胞对破骨细胞分化与骨吸收的抑制作用目的探讨CD4⁺CD25⁺调节性T细胞对人胚骨髓细胞来源的破骨细胞分化与骨吸收的调控作用及其调控机制。方法用免疫磁珠分选法从成人外周血PBMCs分离纯化CD4⁺CD25⁺调节性T细胞。以不同比例同骨髓细胞直接接触共培养与Transwell共培养,观察CD4⁺CD25⁺调节性T细胞对破骨细胞分化与骨吸收作用的影响,并用ELISA检测共培养上清IL-10与TGF-β1水平。在共培养系统中加入抗-IL10与抗-TGFβ1中和抗体,观察调节性T细胞对破骨细胞分化与骨吸收的调控,以解析其调控机制。结果Tregs与BMCs共培养时,Tregs抑制破骨细胞分化与骨吸收作用;Tregs与BMCs比例为1:50时仍具有抑制作用。比较Tregs与BMCs直接接触共培养与Transwell共培养,发现两组间差异无统计学意义（P＞0.05）,表明Tregs主要通过分泌可溶性因子对OC的分化与功能发挥抑制作用。对共培养上清中IL-10与TGF-β1水平分析发现,共培养组IL-10与TGF-β1水平显著高于单独培养BMC组（P＜0.01）,并且随着Tregs与BMC比例的增加,IL-10与TGF-β1的分泌水平增加;比较直接接触共培养与Transwell共培养,发现两者的分泌水平在两组间差异无统计学意义。当共培养系统中联合应用抗-IL10和抗-TGFβ1中和抗体后,能逆转Tregs对OC分化和骨吸收的抑制作用。结论CD4⁺CD25⁺调节性T细胞通过分泌细胞因子的方式抑制破骨细胞分化与骨吸收功能,其中IL-10与TGF-β1具有至关重要的作用。本研究对于理解骨稳态,及类风湿性关节炎、骨质疏松等疾病的发病机制及治疗新策略研究提供了科学依据。第三部分17β—雌二醇通过促进CD4⁺CD25⁺Foxp3⁺调节性T细胞分泌IL-10抑制破骨细胞分化与骨吸收目的探讨17β-E2对CD4⁺CD25⁺Foxp3⁺调节性T细胞与骨髓细胞共培养时对破骨细胞分化与骨吸收的调控以及其作用机制。方法将Tregs/BMCs以2:50的比例直接接触共培养,同时设立未加入Tregs的BMCs组以及未加入BMCs的Tregs组,共3组。三组分别加入不同浓度的E2:10^-7、10^-8、10^-9、10^-10mol/L,并且以相应浓度的溶媒为对照,分别加入有玻片和象牙片的24孔板培养7天和10天,进行TRAP染色和甲苯胺兰染色,并且收集培养48小时的上清,用ELISA检测IL-10与TGF-β1的水平。结果在BMCs组和Tregs/BMCs共培养组加入E2后发现,E2在10^-7-10^-9mol/L的浓度时对OC的分化和骨吸收呈现明显的抑制作用（P＜0.05）;这种抑制作用与E2的浓度呈剂量依赖性,随着E2浓度的降低,这种抑制作用减弱;当E2在10^-10mol/L的浓度时就失去了这种抑制作用（P＞0.05）。而共培养组OC的分化和骨吸收受抑制更显著。通过检测上清中IL-10与TGF-β1的水平,发现在Tregs组与Tregs-BMCs共培养两组中,IL10表达比BMCs组明显增高（P＜0.01）,并且其水平与E2的浓度呈正相关;TGF-β1在Tregs组与Tregs-BMCs共培养中的表达明显高于BMCs组（P＜0.05）,但不受E2浓度的影响。结论17β—雌二醇抑制OC的分化和骨吸收,并且在CD4⁺CD25⁺Foxp3⁺调节性T细胞的作用下,这种抑制作用更为显著,这与E2通过增强Tregs分泌IL-10的作用密切相关。提示IL-10在OC-Tregs这个骨免疫网络中具有至关重要的作用,可以作为PMO治疗的靶点。更多还原

【Abstract】 The immune and skeletal system share a lot of regulatory factors including cytokines and its receptors,signaling molecules and transcription factors;both of the bone and immune cells are in the same micro-environment,bone marrow.Various factors produced and released during immune responses markedly affect bone cells and bone metabolism.Communication between the immune and the skeletal systems is observed in physiological status,but more obviously in autoimmune and other inflammatory diseases.This interrelationship leads to Arron and Choi to propose a concept ’osteoimmunology’ to describe the interaction between immune system and bone metabolism.Osteoclasts play pivotal roles in bone remodeling and some diseases as Postmenopausal osteoporosis（PMO）,rheumatoid arthritis（RA） and periodontal disease.Osteoclasts are multinucleated cells that derive from hematopoietic progenitors in the bone marrow.Receptor activator of nuclear factor kappaB ligand （RANKL） and macrophage colony-stimulating factor（M-CSF） induces the differentiation of hematopoietic progenitors into mature osteoclasts.In RA and inflammatory bowel disease,activated T cells were observed to express RANKL and promote osteoclastogenesis,which links the activation of the immune system and osteoclastogenesis,bone resorption.The present study is to elucidate the mechanisms underlying modulation of CD4⁺CD25⁺Foxp3⁺Regulatory T Cells on Osteoclast differentiation and bone resorption.PartⅠSuppressive regulation of IL-10 and TGF-β1 derived from regulatory T cells on the osteoclast differentiation and bone resorptionObjective Regulatory T cells play an immune regulatory role by secreting such cytokines as IL-10 and TGF-β1.The aim of this study is to investigate the effect of IL-10 and TGF-β1 derived from regulatory T cells on the osteoclast differentiation and bone resorption.Methods Human bone marrow cells were isolated from embryos terminated in 13 and 16 weeks gestation fro non-medicine reason with mifepristone and misoprostol, and maintained in MEM alpha supplemented with 50ng/ml M-CSF and RANKL. After treated with different of concentration of IL-10 or TGF-β1,the BMCs were used for differentiation assay by tartrate-resistant acid phosphatase（TRAP）-staining. Bone resorption was performed in a resorption pit assay using ivory dentine slices with Image-Pro plus 6.0.Results IL-10 or TGF-β1 of 50ng/ml concentration is able to significantly inhibit the osteoclast differentiation and pit formation on dentine slices（P＜0.05）,and the inhibition is in a dosage-dependent manner.The combined treatment of IL-10 and TGF-β1 to BMCs exhibit synergistic effect on osteoclastogenesis and bone resorption at a lower concentration,10ng/ml（P＜0.01）.Conclusion These results above indicate that IL-10 and/or TGF-β1,efficiency cytokines of the regulatory T cells,suppress osteoclastogenesis and bone resorption mediated by the osteoclasts.PartⅡImpact of the CD4⁺CD25⁺Foxp3⁺regulatory T cells on the osteoclast differentiation and bone resorptionObjective The study is to investigate impact and mechanism of the CD4⁺CD25⁺ Foxp3⁺ regulatory T cells on the differentiation of OC from bone marrow cells and bone resorption.Methods The CD4⁺CD25⁺ Foxp3⁺ regulatory T cells were isolated and purified from the PBMCs of healthy adults with MACS.To Study the effect of Tregs on the differentiation of OC from bone marrow cells and bone resorption,the Tregs and BMCs were in direct co-culture or indirect co-culture by transwell in different ratios from 10:50 to 1:50.The IL-10 and TGF-β1 production in the co-culture supematant was determined by ELISA.Moreover,the cytokine-blocking experiments were performed with anti-IL-10 and anti-TGFβ1 antibodies.Results The CD4+^CD25⁺ Foxp3⁺ regulatory T cells inhibit the differentiation of OC from bone marrow cells in a dose-dependent manner（P＜0.05）.Pit formation is also inhibited by Tregs（P＜0.05）.There is no difference of the suppressive effect between direct and indirect co-culture（P＞0.05）.Further study indicates that the inhibition depends on cytokine excretion.IL-10 and TGF-β1 production is up-regulated with the increasing ratio ofTregs:BMCs.After treated with anti-IL-10 and anti-TGFβ1 antibodies,the suppression of Tregs to OC differentiation and bone resorption is reversed completely.Conclusion These data suggest that the CD4⁺CD25⁺ Foxp3⁺ regulatory T cells can suppress osteoclast differentiation and bone resorption by way of secretion of IL-10 and TGF-β1.PartⅢ17-beta estradiol induces CD4⁺CD25⁺Foxp3⁺ regulatory T cells to secret IL-10 for suppression of OC differentiation and bone resorptionObjective The study is to explore the regulation of 17-beta estradiol in the OC differentiation and function mediated by Tregs.Methods Tregs and BMCs were co-cultured at ratio 2:50 with respective BMCs or Tregs as control.The co-culture was treated respectively by different concentrations of E2 from 10^-7 to10^-10 mol/L for 48 hours with dissolvent as control.Thereafter,the supernatant was collected to determine the levels of IL-10 and TGF-β1 with ELISA.After further culture of 7 and 10 days,the TRAP positive cells and bone resorption lacuna area were calculated,respectively.Results The OC differentiation and bone resorption were suppressed efficiently by E2 at the concentration of 10^-7-10^-9mol/L in the BMCs and Tregs/BMCs（P＜0.05）. And the inhibition of E2 on OC differentiation and bone resorption showed a dose-dependent manner.When E2 reached 10^-10 mol/L,it had no significant effect （P＞0.05）.IL-10 and TGF-β1 expression was up-regulated after adding Tregs（P＜0.05）. After treated with E2,the IL-10 production in the Tregs/BMCs and Tregs supernatant shows a pronounced increase（P＜0.01）,but TGF-β1 had no significant change （P＞0.05）.In addition,the secretion of IL-10 increases with the increasing concentration of E2.Conclusion 17-beta estradiol has suppressive effects on OC differentiation and bone resorption.E2 at physiological dosage enhances IL-10 secretion by Tregs,and inhibits OC differentiation and bone resorption.IL-10 is likely involved in the regulation of E2 on bone metabolism via the interaction of Tregs to osteoclast,which may act as potential target for the therapeutics of PMO.更多还原