本文選題：自身免疫 + 免疫調(diào)節(jié)��； 參考：《北京協(xié)和醫(yī)學(xué)院》2015年博士論文

【摘要】：背景：原發(fā)免疫性血小板減少癥(primary immune thrombocytopenia, ITP)是一類由于自身免疫導(dǎo)致的血小板破壞增加、血小板生成受損的疾病。由于自身免疫耐受的打破,從而導(dǎo)致針對血小板以及巨核細(xì)胞的體液免疫和細(xì)胞免疫是慢性ITP的主要發(fā)病機制。間充質(zhì)干細(xì)胞(Mesenchymal stem cells, MSCs)是一類能夠在自身免疫耐受中發(fā)揮免疫調(diào)節(jié)功能的細(xì)胞群體,在多種自身免疫性疾病中有報道骨髓(BM)-MSCs的異常生物學(xué)特征和免疫學(xué)特性。目的：本研究旨在比較研究慢性ITP患者BM-MSCs和正常對照BM-MSCs生物學(xué)特性及功能的差異,為探討慢性ITP患者BM-MSCs是否存在異常提供實驗基礎(chǔ)及理論依據(jù),亦為慢性ITP的發(fā)病機制研究探討新思路。方法：(1)采用貼壁、傳代培養(yǎng)的方法從慢性ITP患者和正常人骨髓組織中獲取相對純化的MSCs,除了采用普通培養(yǎng)基培養(yǎng)外,還設(shè)立了添加bFGF、EGF和ITS這三種額外添加物(addition supplements, AS);(2)倒置顯微鏡觀察BM-MSCs的形態(tài)特征；(3)流式細(xì)胞儀檢測培養(yǎng)的BM-MSCs表面標(biāo)志；(4)定向誘導(dǎo)BM-MSCs向脂肪細(xì)胞和成骨細(xì)胞分化,進行相應(yīng)染色,比較成脂、成骨的分化潛能；(5)測定BM-MSCs增殖能力并繪制生長曲線；(6)流式細(xì)胞儀方法檢測BM-MSCs細(xì)胞周期及細(xì)胞凋亡；(7)建立BM-MSCs與外周單個核細(xì)胞(PBMCs)共培養(yǎng)體系,比較研究BM-MSCs對PHA活化的PBMCs細(xì)胞的增殖、分泌TNF-α、 IFN-γ和IL-10的影響；(8)建立BM-MSCs與CD4+T淋巴細(xì)胞共培養(yǎng)體系,研究BM-MSCs對CD4+T細(xì)胞向CD4+CD25+CD1271ow調(diào)節(jié)性T細(xì)胞的分化的能力。結(jié)果：(1)成功培養(yǎng)并獲取高純度的慢性ITP患者骨髓間充質(zhì)干細(xì)胞；(2)采用普通培養(yǎng)基培養(yǎng),慢性ITP患者BM-MSCs與正常BM-MSCs存在形態(tài)學(xué)差異,正常BM-MSCs細(xì)胞體積小、纖細(xì)、呈長梭形,規(guī)則排列生長；ITP患者BM-MSCs細(xì)胞扁平且體積較大,呈短梭形、多角形、不規(guī)則形,邊緣不整齊。AS能夠改善ITP患者BM-MSCs形態(tài)異常,與正常BM-MSCs無明顯差別。(3)ITP患者BM-MSCs與正常對照BM-MSCs均表達CD73、CD105和CD90,均不表達造血細(xì)胞標(biāo)志CD34、CD45,不表達CDllb、CD14和CDl9。(4)ITP患者BM-MSCs分化為脂肪細(xì)胞和成骨細(xì)胞能力與正常BM-MSCs相比未見明顯差異。(5)ITP患者BM-MSCs細(xì)胞凋亡水平明顯高于正常對照。AS處理能夠減少BM-MSCs的凋亡水平,此時ITP患者同正常對照無明顯差異。(7)ITp患者BM-MSCs以劑量依賴的方式抑制正常來源PBMCs增殖,但與正常BM-MSCs相比能力下降。ITP患者BM-MSCs抑制PBMCs對IFN-γ、TNF-α的分泌,但是促進PBMCs分泌IL-10的能力相比正常BM-MSCs下降。(8)ITP患者BM-MSCs誘導(dǎo)正常來源CD4+ T細(xì)胞分化為Treg的能力與正常BM-MSCs相比降低。結(jié)論：慢性ITP患者來源的BM-MSCs增殖能力受損,形態(tài)異常以及過度凋亡,而這些缺陷可以被改良的培養(yǎng)條件矯正。慢性ITP患者BM-MSCs與正常來源的BM-MSCs相比有著相同的免疫表型和相似的分化成骨成脂能力。然而慢性ITP患者來源的BM-MSCs的免疫抑制功能和誘導(dǎo)Tregs的能力相比正常來源的BM-MSCs明顯下降。這些研究發(fā)現(xiàn)提示了慢性ITP患者BM-MSCs存在一定缺陷,可能與慢性ITP的發(fā)病相關(guān)。背景：原發(fā)性免疫性血小板減少癥(primary immune thrombocytopenia, ITP)是一種器官特異性的自身免疫性疾病,伴隨多種免疫異常,包括自身反應(yīng)性淋巴細(xì)胞的過度增殖和凋亡抵抗。白介素7 (Interleukin-7, IL-7)是由非骨髓來源的間質(zhì)細(xì)胞和內(nèi)皮細(xì)胞產(chǎn)生,能夠維持靜息CD4+T細(xì)胞和CD8+T細(xì)胞穩(wěn)態(tài)的一類重要細(xì)胞因子,在T細(xì)胞清除時血漿以及組織中的IL-7水平會明顯升高。而且IL-7也能夠維持未成熟B細(xì)胞的存活并在免疫球蛋白的產(chǎn)生中具有重要作用。目的：本實驗主要目的是想確定IL-7對ITP患者外周單個核細(xì)胞(peripheral blood mononuclear cells, PBMCs)和骨髓單個核細(xì)胞(bone marrow mononuclear cells, BMNCs)的作用。方法：(1)通過ELISA方法檢測血漿及細(xì)胞培養(yǎng)上清中細(xì)胞因子的表達；(2)通過流式細(xì)胞術(shù)檢測CD4+細(xì)胞表面IL-7α的表達；(3)通過BrdU摻入的方法檢測IL-7對PBMCs和BMNCs細(xì)胞增殖的作用；(4)通過流式細(xì)胞術(shù)檢測IL-7對血小板、PBMCs和BMNCs的凋亡影響。結(jié)果：通過ELISA檢測,我們發(fā)現(xiàn)ITP患者外周血血漿IL-7水平低于正常對照,與是否用藥無關(guān),與患者血小板水平正相關(guān)；疾病緩解后IL-7表達升高但仍然低于正常對照。骨髓血漿中IL-7在ITP與患者之間并沒有明顯區(qū)別。ITP患者和正常對照CD4+T細(xì)胞表面IL-7Rα表達水平?jīng)]有差異。體外刺激實驗顯示IL-7上調(diào)了ITP患者自體血小板的凋亡,促進外周以及骨髓單個核細(xì)胞的增殖,促進了IFN-γ、TNF-α和IL-10的分泌,抑制CD8+T淋巴細(xì)胞的凋亡。結(jié)論：ITP患者體內(nèi)IL-7水平降低,同時對于淋巴細(xì)胞的作用同正常對照相比存在異常。由于IL-7能夠促進淋巴細(xì)胞凋亡耐受并且具有促炎作用,我們推測IL-7在ITP患者血漿水平的降低可能是由于促炎作用的負(fù)反饋調(diào)節(jié)機制引起。
[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 - 偽 , IFN - 緯 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偽 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 - 緯 , TNF - 偽 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 .
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R558.2

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1 Justin D Glenn;Katharine A Whartenby;;Mesenchymal stem cells: Emerging mechanisms of immunomodulation and therapy[J];World Journal of Stem Cells;2014年05期

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本文編號：1998627