RNA干擾抑制樹(shù)突細(xì)胞CD70在免疫性血小板減少癥發(fā)病機(jī)制中的研究
發(fā)布時(shí)間：2018-09-19 11:09
【摘要】：[目的]慢性原發(fā)性免疫血小板減少癥(ITP)是一種自身免疫性疾病,其主要表現(xiàn)為血小板減少和出血風(fēng)險(xiǎn)增加。其發(fā)病機(jī)制還不完全清楚,體液免疫和細(xì)胞免疫是主要的發(fā)病機(jī)制。樹(shù)突狀細(xì)胞(DCs)是誘導(dǎo)初級(jí)免疫應(yīng)答的抗原呈遞細(xì)胞。DC不僅在誘導(dǎo)初級(jí)免疫應(yīng)答中起關(guān)鍵作用,而且對(duì)于誘導(dǎo)免疫耐受和調(diào)節(jié)T細(xì)胞介導(dǎo)的免疫應(yīng)答也起重要作用,在一些自身免疫性疾病中DC表現(xiàn)出異常的免疫學(xué)特征。本研究探討慢性ITP患者DCs和正常人DCs上共刺激分子CD70的生物學(xué)特性及功能的差異,以明確CD70在慢性ITP患者發(fā)病機(jī)制中的作用,進(jìn)一步闡明慢性ITP患者的自身免疫學(xué)發(fā)病機(jī)制。[方法](1)選取18例慢性ITP患者,病程超過(guò)12個(gè)月,分離外周血單個(gè)核細(xì)胞。(2)貼壁法體外刺激單核系DCs細(xì)胞,流式細(xì)胞儀鑒定DCs細(xì)胞表面標(biāo)志。(3)流式和Rt-PCR的方法篩選合成的3條siRNA中干擾CD70效果最好的一條。(4)脂質(zhì)體介導(dǎo)下轉(zhuǎn)染DCs細(xì)胞,檢測(cè)DC細(xì)胞CD70mRNA的表達(dá)和蛋白的表達(dá)。(5)混合淋巴細(xì)胞共培養(yǎng),轉(zhuǎn)染siRNA的DCs和轉(zhuǎn)染陰性對(duì)照DCs分別與CD4+T細(xì)胞共培養(yǎng),檢測(cè)DCs對(duì)CD4+T細(xì)胞增殖的影響,以及在TGF-β誘導(dǎo)下DCs對(duì)CD4+T細(xì)胞向CD4+CD25+CD127-調(diào)節(jié)T細(xì)胞的分化能力。(6)檢測(cè)共培養(yǎng)體系的培養(yǎng)上清中分泌IFN-γ、IL-10影響。[結(jié)果](1)ITP患者的DCs與正常人DCs相比,樹(shù)突更為明顯和密集,表面標(biāo)志無(wú)明顯差異,ITP 患者 CDla(55.5±1.6)%,CD11c(77.7±0.6)%,CD86(75.1±4.0)%,HLA-DR(83.7±2.8)%。(2)經(jīng)脂質(zhì)體轉(zhuǎn)染4-6小時(shí)后,檢測(cè)轉(zhuǎn)染率為60%左右。(3)siRNA1\2\3轉(zhuǎn)染293T細(xì)胞后,48小時(shí)測(cè)CD70mRNA的表達(dá)明顯減少,抑制率分別為(63.6±8.5)%,(69.0±11.7)%,(86.1 ±3.2)%。而未轉(zhuǎn)染組和陰性對(duì)照組對(duì)CD70mRNA表達(dá)均無(wú)統(tǒng)計(jì)學(xué)差異。(4)siRNA3轉(zhuǎn)染DCs,轉(zhuǎn)染siRNA3組CD70mRNA明顯低于對(duì)照組,CD70蛋白表達(dá)也明顯低于對(duì)照組,未轉(zhuǎn)染組和陰性對(duì)照組CD70蛋白的表達(dá)無(wú)明顯差異。(5)PHA可以明顯促進(jìn)ITP患者DCs刺激CD4+T細(xì)胞增殖,ITP患者DCs轉(zhuǎn)染siRNA3組的增殖低于陰性對(duì)照組,正常人DCs轉(zhuǎn)染siRNA3組相對(duì)于轉(zhuǎn)染陰性對(duì)照組無(wú)明顯差異。(6)ITP患者和正常人轉(zhuǎn)染siRNA3組分泌IFN-y均低于轉(zhuǎn)染陰性對(duì)照組,分泌IL-10均高于陰性對(duì)照組。(7)在TGF-β誘導(dǎo)下,ITP患者和正常DCs轉(zhuǎn)染siRNA3誘導(dǎo)正常來(lái)源CD4+T細(xì)胞分化的Treg細(xì)胞均低于轉(zhuǎn)染陰性對(duì)照組。[結(jié)論]ITP患者的DCs比正常對(duì)照組更密集,可能是ITP患者中DCs相對(duì)于正常人功能亢進(jìn)。siRNA可特異抑制樹(shù)突細(xì)胞CD70基因的轉(zhuǎn)錄和表達(dá)。用siRNA干擾DC細(xì)胞CD70后抑制了 CD4+T細(xì)胞增殖,誘導(dǎo)分化Treg細(xì)胞功能減弱,逆轉(zhuǎn)了 ITP患者Th1/Th2的極化狀態(tài)。本研究結(jié)果為自身免疫疾病提供了新思路和治療途徑。[目的]原發(fā)性免疫血小板減少癥(ITP)是一種自身免疫性疾病,其主要表現(xiàn)為血小板減少和出血風(fēng)險(xiǎn)增加。盡管CD83是成熟樹(shù)突狀細(xì)胞(DCs)的選擇性標(biāo)記物,但也表達(dá)于多種其他細(xì)胞,包括胸腺上皮細(xì)胞,T細(xì)胞,特別是調(diào)節(jié)性T細(xì)胞和B細(xì)胞。CD83和sCD83可能在自身免疫中發(fā)揮重要作用。在我們的研究中,我們研究ITP患者CD4+T細(xì)胞上的表達(dá),以及經(jīng)典的CD3/CD28信號(hào)和轉(zhuǎn)化生長(zhǎng)因子(TGF)-β刺激對(duì)CD4+T細(xì)胞CD83表達(dá)的影響以及它們?cè)贗TP中分化成CD4+CD25+FoxP3+誘導(dǎo)的調(diào)節(jié)性T(iTreg)細(xì)胞的研究。旨在探討ITP患者CD4+T細(xì)胞亞群上CD83表達(dá)的情況以及與ITP患者病情和預(yù)后的相關(guān)性。[方法](1)76例ITP患者入組,均符合2009年國(guó)際專家共識(shí)的診斷標(biāo)準(zhǔn),排除其他自身免疫病。(2)通過(guò)流式細(xì)胞儀檢測(cè)ITP患者和正常人CD83+CD4+T呈現(xiàn)時(shí)間依賴性關(guān)系;不同分組的ITP患者與正常對(duì)照組檢測(cè)各組細(xì)胞群的CD83+CD4+T細(xì)胞的表達(dá)情況以及與性別,年齡,病程,血小板計(jì)數(shù)等臨床指標(biāo)之間的相關(guān)性分析;(3)ITP患者和健康人外周血單個(gè)核細(xì)胞(PBMCs)體外培養(yǎng),培養(yǎng)體系中分別加入 anti-CD3/28;anti-CD3/28+TGF-β;anti-CD3/28+TGF-β +CD83單克隆抗體,通過(guò)實(shí)時(shí)定量聚合酶鏈反應(yīng)檢測(cè)PBMCs中CD83的mRNA表達(dá)水平,流式細(xì)胞儀檢測(cè)CD83的表達(dá)與CD4+CD25-、CD4+CD25+、CD4+CD25+FOXp3+Treg的關(guān)系。(4)酶聯(lián)免疫吸附測(cè)定血漿中sCD83濃度,sCD83與血小板數(shù)的相關(guān)性分析。[結(jié)果]1.不同分組間CD83+CD4+T細(xì)胞表現(xiàn)為ITP患者初治組、活動(dòng)期、緩解組和正常對(duì)照組分別為(6.47%±0.55%);(3.93%±0.46%);(2.97%±0.26%);(2.60%±0.33),初治組未用藥組和活動(dòng)期顯著高于緩解組和正常對(duì)照組,差異均有統(tǒng)計(jì)學(xué)意義(P均0.01)。2.ITP患者和正常人外周血單個(gè)核細(xì)胞在抗CD3/CD28刺激下,CD83+CD4+T細(xì)胞表現(xiàn)出時(shí)間依賴性,ITP患者組在0天時(shí)CD83+CD4+T表達(dá)很低,而在第一天時(shí)CD83+CD4+T陽(yáng)性的百分比開(kāi)始上調(diào)(2.49±0.31%),第2天進(jìn)一步顯著增加(5.48±0.48%),到第三天開(kāi)始降低至(1.97±0.32%)。3.在ITP患者組和正常人組的CD4+CD25-細(xì)胞CD83表達(dá)均較低,CD4+CD25+細(xì)胞CD83表達(dá)較高,CD4+CD25+FOXp3+Treg細(xì)胞CD83表達(dá)最高4.antiCD83下調(diào)CD83的表達(dá),TGF-β上調(diào)CD83的表達(dá)。ITP患者組CD83分子mRNA的相對(duì)表達(dá)量顯著高于正常對(duì)照組。ITP患者外周血漿中sCD83水平升高,與CD83+CD4+T表達(dá)相一致,并與血小板數(shù)目呈負(fù)相關(guān)(r=-0.438,p=0.007),而與性別和年齡無(wú)相關(guān)性。[結(jié)論]CD83+CD4+T細(xì)胞與ITP患者病程呈顯著負(fù)相關(guān)。血小板計(jì)數(shù)與血漿sCD83水平呈顯著負(fù)相關(guān)。隨著病情的緩解,CD83+CD4+T細(xì)胞表達(dá)和血漿sCD83水平明顯下調(diào)。CD83在Treg上表達(dá)升高可能與其免疫抑制功能異常有關(guān),同時(shí)sCD83的水平未來(lái)可能可用來(lái)監(jiān)測(cè)ITP患者病情變化。
[Abstract]:[Objective] Chronic idiopathic immune thrombocytopenia (ITP) is an autoimmune disease characterized by thrombocytopenia and increased risk of bleeding. Its pathogenesis is not fully understood. Humoral and cellular immunity are the main pathogenesis. Dendritic cells (DCs) are antigen presenting cells that induce primary immune responses. It plays a key role in inducing primary immune response, and it also plays an important role in inducing immune tolerance and regulating T cell mediated immune response. DC shows abnormal immunological characteristics in some autoimmune diseases. This study was to investigate the biological characteristics and function of CD70 co-stimulatory molecule on DCs of chronic ITP patients and normal subjects. To clarify the role of CD70 in the pathogenesis of chronic ITP patients, and further elucidate the autoimmune pathogenesis of chronic ITP patients. [Methods] (1) 18 patients with chronic ITP were selected and their peripheral blood mononuclear cells were isolated over 12 months. (2) Mononuclear DCs cells were stimulated in vitro by adherence method and identified by flow cytometry. (4) Liposome-mediated transfection of DCs to detect the expression of CD70 mRNA and protein. (5) Co-culture of mixed lymphocytes, co-culture of the DCs transfected with siRNA and C D4 + T cells transfected with negative control DCs, and detection of C D4 + T cells by DCs. The effect of DCs on the proliferation of CD4+T cells and the ability of DCs to differentiate into CD4+CD25+CD127-regulated T cells induced by TGF-beta were investigated. (6) The secretion of IFN-gamma and IL-10 in the supernatant of co-culture system was detected. [Results] (1) DCs from ITP patients were more dendritic and dense than normal DCs, and the surface markers of CDla (55) in ITP patients were not significantly different. (2) After 4-6 hours of lipofectin transfection, the transfection rate was about 60%. (3) After transfection with siRNA 123 into 293T cells, the expression of CD70 mRNA was significantly decreased at 48 hours, and the inhibition rates were (63.6 (+) 8.5)%, (69.0 (+) 11.7)%, (86.1 (+) 3.2)%. There was no significant difference in the expression of NA. (4) The expression of CD70 mRNA and CD70 protein in siRNA 3 transfected DCs was significantly lower than that in control group, and there was no significant difference in the expression of CD70 protein between non-transfected group and negative control group. (5) PHA could significantly promote the proliferation of CD4 + T cells in ITP patients, and the proliferation of DCs transfected with siRNA 3 was lower in ITP patients. In the negative control group, there was no significant difference in the secretion of IFN-y and IL-10 between the normal and ITP-transfected siRNA-3 groups. (6) The secretion of IFN-y was lower than that of the negative control group, and IL-10 was higher than that of the negative control group. (7) Under the induction of TGF-beta, the differentiation of normal CD4 + T cells was induced by the transfection of siRNA-3 into ITP patients and normal DCs. [Conclusion] DCs in ITP patients are more dense than those in normal controls, which may be due to the hyperfunction of DCs in ITP patients. SiRNA can specifically inhibit the transcription and expression of CD70 gene in dendritic cells. [Objective] Primary immune thrombocytopenia (ITP) is an autoimmune disease characterized by thrombocytopenia and increased risk of bleeding. Although CD83 is a selective form of mature dendritic cells (DCs), CD83 is an autoimmune disease. CD83 and sCD83 may play an important role in autoimmunity. In our study, we investigated the expression of CD4 + T cells in ITP patients, as well as classical CD3 / CD28 signaling and transforming growth factor (TGF) - beta stimulation. The purpose of this study was to investigate the expression of CD83 in CD4 + T cells and their differentiation into CD4 + CD25 + FoxP3 + induced regulatory T (iTreg) cells in ITP. The purpose of this study was to investigate the relationship between CD83 expression in CD4 + T cell subsets and the condition and prognosis of ITP patients. The expression of CD83 + CD4 + T cells in different groups of ITP patients and normal controls, and the correlation between CD83 + CD4 + T cell expression and clinical parameters such as sex, age, course of disease, platelet count were detected. Analysis; (3) Peripheral blood mononuclear cells (PBMCs) from patients with ITP and healthy persons were cultured in vitro. Anti-CD3/28, anti-CD3/28+TGF-beta, anti-CD3/28+TGF-beta and anti-CD83 monoclonal antibodies were added to the culture system. The expression of CD83 mRNA in PBMCs was detected by real-time quantitative polymerase chain reaction, and the expression of CD83 was detected by flow cytometry. The relationship between D4+CD25+, CD4+CD25+FOXp3+Treg. (4) The concentration of sCD83 in plasma was measured by enzyme-linked immunosorbent assay, and the correlation between sCD83 and platelet count was analyzed. [Results] 1. CD83+CD4+T cells in different groups were found in the initial treatment group, active stage, remission group and normal control group (6.47%+0.55%); (3.93%+0.46%); (2.97%+0.26%); (2.60%+0.33), respectively. The expression of CD83+CD4+T cells in the untreated group was significantly higher than that in the remission group and the normal control group (P 0.01). The percentage of sex began to increase (2.49 [0.31]%) and further increased (5.48 [0.48]%) on the second day, then decreased to (1.97 [0.32]%) on the third day. The expression of CD83 in CD4 + CD25 - cells was lower in patients with ITP and normal subjects, the expression of CD83 in CD4 + CD25 + cells was higher, the expression of CD4 + CD25 + FOXp3 + Treg cells was lowered by 4.CD83, and TG was lowered by 4.CD83. The relative expression of CD83 mRNA in ITP patients was significantly higher than that in normal controls. The expression of sCD83 in peripheral plasma was consistent with that of CD83+CD4+T, and was negatively correlated with the number of platelets (r=-0.438, p=0.007), but not with sex and age. Platelet counts were negatively correlated with plasma sCD83 levels. The expression of CD83 + CD4 + T cells and plasma sCD83 levels were significantly down-regulated with remission. The increased expression of CD83 on Treg may be associated with abnormal immunosuppressive function, and the level of sCD83 may be used to monitor the changes of ITP patients in the future.
【學(xué)位授予單位】：昆明醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R558.2
本文編號(hào)：2249956
[Abstract]:[Objective] Chronic idiopathic immune thrombocytopenia (ITP) is an autoimmune disease characterized by thrombocytopenia and increased risk of bleeding. Its pathogenesis is not fully understood. Humoral and cellular immunity are the main pathogenesis. Dendritic cells (DCs) are antigen presenting cells that induce primary immune responses. It plays a key role in inducing primary immune response, and it also plays an important role in inducing immune tolerance and regulating T cell mediated immune response. DC shows abnormal immunological characteristics in some autoimmune diseases. This study was to investigate the biological characteristics and function of CD70 co-stimulatory molecule on DCs of chronic ITP patients and normal subjects. To clarify the role of CD70 in the pathogenesis of chronic ITP patients, and further elucidate the autoimmune pathogenesis of chronic ITP patients. [Methods] (1) 18 patients with chronic ITP were selected and their peripheral blood mononuclear cells were isolated over 12 months. (2) Mononuclear DCs cells were stimulated in vitro by adherence method and identified by flow cytometry. (4) Liposome-mediated transfection of DCs to detect the expression of CD70 mRNA and protein. (5) Co-culture of mixed lymphocytes, co-culture of the DCs transfected with siRNA and C D4 + T cells transfected with negative control DCs, and detection of C D4 + T cells by DCs. The effect of DCs on the proliferation of CD4+T cells and the ability of DCs to differentiate into CD4+CD25+CD127-regulated T cells induced by TGF-beta were investigated. (6) The secretion of IFN-gamma and IL-10 in the supernatant of co-culture system was detected. [Results] (1) DCs from ITP patients were more dendritic and dense than normal DCs, and the surface markers of CDla (55) in ITP patients were not significantly different. (2) After 4-6 hours of lipofectin transfection, the transfection rate was about 60%. (3) After transfection with siRNA 123 into 293T cells, the expression of CD70 mRNA was significantly decreased at 48 hours, and the inhibition rates were (63.6 (+) 8.5)%, (69.0 (+) 11.7)%, (86.1 (+) 3.2)%. There was no significant difference in the expression of NA. (4) The expression of CD70 mRNA and CD70 protein in siRNA 3 transfected DCs was significantly lower than that in control group, and there was no significant difference in the expression of CD70 protein between non-transfected group and negative control group. (5) PHA could significantly promote the proliferation of CD4 + T cells in ITP patients, and the proliferation of DCs transfected with siRNA 3 was lower in ITP patients. In the negative control group, there was no significant difference in the secretion of IFN-y and IL-10 between the normal and ITP-transfected siRNA-3 groups. (6) The secretion of IFN-y was lower than that of the negative control group, and IL-10 was higher than that of the negative control group. (7) Under the induction of TGF-beta, the differentiation of normal CD4 + T cells was induced by the transfection of siRNA-3 into ITP patients and normal DCs. [Conclusion] DCs in ITP patients are more dense than those in normal controls, which may be due to the hyperfunction of DCs in ITP patients. SiRNA can specifically inhibit the transcription and expression of CD70 gene in dendritic cells. [Objective] Primary immune thrombocytopenia (ITP) is an autoimmune disease characterized by thrombocytopenia and increased risk of bleeding. Although CD83 is a selective form of mature dendritic cells (DCs), CD83 is an autoimmune disease. CD83 and sCD83 may play an important role in autoimmunity. In our study, we investigated the expression of CD4 + T cells in ITP patients, as well as classical CD3 / CD28 signaling and transforming growth factor (TGF) - beta stimulation. The purpose of this study was to investigate the expression of CD83 in CD4 + T cells and their differentiation into CD4 + CD25 + FoxP3 + induced regulatory T (iTreg) cells in ITP. The purpose of this study was to investigate the relationship between CD83 expression in CD4 + T cell subsets and the condition and prognosis of ITP patients. The expression of CD83 + CD4 + T cells in different groups of ITP patients and normal controls, and the correlation between CD83 + CD4 + T cell expression and clinical parameters such as sex, age, course of disease, platelet count were detected. Analysis; (3) Peripheral blood mononuclear cells (PBMCs) from patients with ITP and healthy persons were cultured in vitro. Anti-CD3/28, anti-CD3/28+TGF-beta, anti-CD3/28+TGF-beta and anti-CD83 monoclonal antibodies were added to the culture system. The expression of CD83 mRNA in PBMCs was detected by real-time quantitative polymerase chain reaction, and the expression of CD83 was detected by flow cytometry. The relationship between D4+CD25+, CD4+CD25+FOXp3+Treg. (4) The concentration of sCD83 in plasma was measured by enzyme-linked immunosorbent assay, and the correlation between sCD83 and platelet count was analyzed. [Results] 1. CD83+CD4+T cells in different groups were found in the initial treatment group, active stage, remission group and normal control group (6.47%+0.55%); (3.93%+0.46%); (2.97%+0.26%); (2.60%+0.33), respectively. The expression of CD83+CD4+T cells in the untreated group was significantly higher than that in the remission group and the normal control group (P 0.01). The percentage of sex began to increase (2.49 [0.31]%) and further increased (5.48 [0.48]%) on the second day, then decreased to (1.97 [0.32]%) on the third day. The expression of CD83 in CD4 + CD25 - cells was lower in patients with ITP and normal subjects, the expression of CD83 in CD4 + CD25 + cells was higher, the expression of CD4 + CD25 + FOXp3 + Treg cells was lowered by 4.CD83, and TG was lowered by 4.CD83. The relative expression of CD83 mRNA in ITP patients was significantly higher than that in normal controls. The expression of sCD83 in peripheral plasma was consistent with that of CD83+CD4+T, and was negatively correlated with the number of platelets (r=-0.438, p=0.007), but not with sex and age. Platelet counts were negatively correlated with plasma sCD83 levels. The expression of CD83 + CD4 + T cells and plasma sCD83 levels were significantly down-regulated with remission. The increased expression of CD83 on Treg may be associated with abnormal immunosuppressive function, and the level of sCD83 may be used to monitor the changes of ITP patients in the future.
【學(xué)位授予單位】：昆明醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R558.2
【參考文獻(xiàn)】
相關(guān)期刊論文 前3條
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2 齊暉,李富榮,劉冬舟,肖學(xué)呂,任莉莉,文錦麗,黃瑞芳;SLE患者外周血DC的表面標(biāo)志及其分泌IL-12和IFN-α的研究[J];細(xì)胞與分子免疫學(xué)雜志;2005年04期
3 ;The Expression and Distribution of S-100 Protein and CD83 in Thyroid Tissues of Autoimmune Thyroid Diseases[J];Cellular & Molecular Immunology;2004年05期
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