骨髓间充质干细胞在免疫性血小板减少症发病机制中的作用研宄

发布时间：2018-06-09 03:20

本文选题：自身免疫 + 免疫调节　；参考：《北京协和医学院》2015年博士论文

【摘要】：背景：原发免疫性血小板减少症(primary immune thrombocytopenia, ITP)是一类由于自身免疫导致的血小板破坏增加、血小板生成受损的疾病。由于自身免疫耐受的打破,从而导致针对血小板以及巨核细胞的体液免疫和细胞免疫是慢性ITP的主要发病机制。间充质干细胞(Mesenchymal stem cells, MSCs)是一类能够在自身免疫耐受中发挥免疫调节功能的细胞群体,在多种自身免疫性疾病中有报道骨髓(BM)-MSCs的异常生物学特征和免疫学特性。目的：本研究旨在比较研究慢性ITP患者BM-MSCs和正常对照BM-MSCs生物学特性及功能的差异,为探讨慢性ITP患者BM-MSCs是否存在异常提供实验基础及理论依据,亦为慢性ITP的发病机制研究探讨新思路。方法：(1)采用贴壁、传代培养的方法从慢性ITP患者和正常人骨髓组织中获取相对纯化的MSCs,除了采用普通培养基培养外,还设立了添加bFGF、EGF和ITS这三种额外添加物(addition supplements, AS);(2)倒置显微镜观察BM-MSCs的形态特征；(3)流式细胞仪检测培养的BM-MSCs表面标志；(4)定向诱导BM-MSCs向脂肪细胞和成骨细胞分化,进行相应染色,比较成脂、成骨的分化潜能；(5)测定BM-MSCs增殖能力并绘制生长曲线；(6)流式细胞仪方法检测BM-MSCs细胞周期及细胞凋亡；(7)建立BM-MSCs与外周单个核细胞(PBMCs)共培养体系,比较研究BM-MSCs对PHA活化的PBMCs细胞的增殖、分泌TNF-α、 IFN-γ和IL-10的影响；(8)建立BM-MSCs与CD4+T淋巴细胞共培养体系,研究BM-MSCs对CD4+T细胞向CD4+CD25+CD1271ow调节性T细胞的分化的能力。结果：(1)成功培养并获取高纯度的慢性ITP患者骨髓间充质干细胞；(2)采用普通培养基培养,慢性ITP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；ITP患者BM-MSCs细胞扁平且体积较大,呈短梭形、多角形、不规则形,边缘不整齐。AS能够改善ITP患者BM-MSCs形态异常,与正常BM-MSCs无明显差别。(3)ITP患者BM-MSCs与正常对照BM-MSCs均表达CD73、CD105和CD90,均不表达造血细胞标志CD34、CD45,不表达CDllb、CD14和CDl9。(4)ITP患者BM-MSCs分化为脂肪细胞和成骨细胞能力与正常BM-MSCs相比未见明显差异。(5)ITP患者BM-MSCs细胞凋亡水平明显高于正常对照。AS处理能够减少BM-MSCs的凋亡水平,此时ITP患者同正常对照无明显差异。(7)ITp患者BM-MSCs以剂量依赖的方式抑制正常来源PBMCs增殖,但与正常BM-MSCs相比能力下降。ITP患者BM-MSCs抑制PBMCs对IFN-γ、TNF-α的分泌,但是促进PBMCs分泌IL-10的能力相比正常BM-MSCs下降。(8)ITP患者BM-MSCs诱导正常来源CD4+ T细胞分化为Treg的能力与正常BM-MSCs相比降低。结论：慢性ITP患者来源的BM-MSCs增殖能力受损,形态异常以及过度凋亡,而这些缺陷可以被改良的培养条件矫正。慢性ITP患者BM-MSCs与正常来源的BM-MSCs相比有着相同的免疫表型和相似的分化成骨成脂能力。然而慢性ITP患者来源的BM-MSCs的免疫抑制功能和诱导Tregs的能力相比正常来源的BM-MSCs明显下降。这些研究发现提示了慢性ITP患者BM-MSCs存在一定缺陷,可能与慢性ITP的发病相关。背景：原发性免疫性血小板减少症(primary immune thrombocytopenia, ITP)是一种器官特异性的自身免疫性疾病,伴随多种免疫异常,包括自身反应性淋巴细胞的过度增殖和凋亡抵抗。白介素7 (Interleukin-7, IL-7)是由非骨髓来源的间质细胞和内皮细胞产生,能够维持静息CD4+T细胞和CD8+T细胞稳态的一类重要细胞因子,在T细胞清除时血浆以及组织中的IL-7水平会明显升高。而且IL-7也能够维持未成熟B细胞的存活并在免疫球蛋白的产生中具有重要作用。目的：本实验主要目的是想确定IL-7对ITP患者外周单个核细胞(peripheral blood mononuclear cells, PBMCs)和骨髓单个核细胞(bone marrow mononuclear cells, BMNCs)的作用。方法：(1)通过ELISA方法检测血浆及细胞培养上清中细胞因子的表达；(2)通过流式细胞术检测CD4+细胞表面IL-7α的表达；(3)通过BrdU掺入的方法检测IL-7对PBMCs和BMNCs细胞增殖的作用；(4)通过流式细胞术检测IL-7对血小板、PBMCs和BMNCs的凋亡影响。结果：通过ELISA检测,我们发现ITP患者外周血血浆IL-7水平低于正常对照,与是否用药无关,与患者血小板水平正相关；疾病缓解后IL-7表达升高但仍然低于正常对照。骨髓血浆中IL-7在ITP与患者之间并没有明显区别。ITP患者和正常对照CD4+T细胞表面IL-7Rα表达水平没有差异。体外刺激实验显示IL-7上调了ITP患者自体血小板的凋亡,促进外周以及骨髓单个核细胞的增殖,促进了IFN-γ、TNF-α和IL-10的分泌,抑制CD8+T淋巴细胞的凋亡。结论：ITP患者体内IL-7水平降低,同时对于淋巴细胞的作用同正常对照相比存在异常。由于IL-7能够促进淋巴细胞凋亡耐受并且具有促炎作用,我们推测IL-7在ITP患者血浆水平的降低可能是由于促炎作用的负反馈调节机制引起。
[Abstract]:BACKGROUND : Primary immune thrombocytopenia ( ITP ) is a kind of disease caused by autoimmunity . It is the main pathogenesis of chronic ITP .
( 3 ) the surface markers of BM - MSCs were detected by flow cytometry ;
( 4 ) directionally inducing BM - MSCs to differentiate into adipocytes and osteoblasts , and performing corresponding staining to compare the differentiation potential of fat and osteogenic differentiation ;
( 5 ) measuring BM - MSCs proliferation ability and drawing growth curve ;
( 6 ) Flow cytometry was used to detect the cell cycle and apoptosis of BM - MSCs .
( 7 ) establishing a co - culture system of BM - MSCs and peripheral mononuclear cells ( PBMCs ) , and comparing the effects of BM - MSCs on proliferation and secretion of TNF - 伪 , IFN - 纬 and IL - 10 by PHA activated PBMCs ;
( 8 ) To establish a co - culture system of BM - MSCs and CD4 + T lymphocytes , and to investigate the ability of BM - MSCs to differentiate CD4 + CD25 + CD1271ow regulatory T cells .
( 2 ) BM - MSCs were cultured in common culture medium . BM - MSCs and normal BM - MSCs had morphological differences .
( 3 ) The BM - MSCs in ITP patients had no significant difference compared with normal BM - MSCs . ( 3 ) The BM - MSCs in ITP patients had no significant difference compared with normal BM - MSCs . ( 8 ) The ability of BM - MSCs in ITP patients to differentiate into Treg is lower than that of normal BM - MSCs .
( 2 ) detecting the expression of IL - 7伪 on CD4 + cell surface by flow cytometry ;
( 3 ) The effect of IL - 7 on the proliferation of PBMCs and BMSC cells was detected by means of the method of incorporation .
( 4 ) IL - 7 was detected by flow cytometry . Results : The level of IL - 7 in peripheral blood of ITP patients was lower than that of normal controls .
The levels of IL - 7 in bone marrow plasma were not significantly different between ITP and patients . The results showed that IL - 7 increased the level of IL - 7 in ITP patients and promoted the proliferation of IFN - 纬 , TNF - 伪 and IL - 10 . Conclusion : The decrease of IL - 7 in ITP patients may be caused by the negative feedback regulation mechanism of pro - inflammatory effect .
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R558.2

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