RNA干扰抑制树突细胞CD70在免疫性血小板减少症发病机制中的研究
发布时间：2018-09-19 11:09
【摘要】：[目的]慢性原发性免疫血小板减少症(ITP)是一种自身免疫性疾病,其主要表现为血小板减少和出血风险增加。其发病机制还不完全清楚,体液免疫和细胞免疫是主要的发病机制。树突状细胞(DCs)是诱导初级免疫应答的抗原呈递细胞。DC不仅在诱导初级免疫应答中起关键作用,而且对于诱导免疫耐受和调节T细胞介导的免疫应答也起重要作用,在一些自身免疫性疾病中DC表现出异常的免疫学特征。本研究探讨慢性ITP患者DCs和正常人DCs上共刺激分子CD70的生物学特性及功能的差异,以明确CD70在慢性ITP患者发病机制中的作用,进一步阐明慢性ITP患者的自身免疫学发病机制。[方法](1)选取18例慢性ITP患者,病程超过12个月,分离外周血单个核细胞。(2)贴壁法体外刺激单核系DCs细胞,流式细胞仪鉴定DCs细胞表面标志。(3)流式和Rt-PCR的方法筛选合成的3条siRNA中干扰CD70效果最好的一条。(4)脂质体介导下转染DCs细胞,检测DC细胞CD70mRNA的表达和蛋白的表达。(5)混合淋巴细胞共培养,转染siRNA的DCs和转染阴性对照DCs分别与CD4+T细胞共培养,检测DCs对CD4+T细胞增殖的影响,以及在TGF-β诱导下DCs对CD4+T细胞向CD4+CD25+CD127-调节T细胞的分化能力。(6)检测共培养体系的培养上清中分泌IFN-γ、IL-10影响。[结果](1)ITP患者的DCs与正常人DCs相比,树突更为明显和密集,表面标志无明显差异,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)经脂质体转染4-6小时后,检测转染率为60%左右。(3)siRNA1\2\3转染293T细胞后,48小时测CD70mRNA的表达明显减少,抑制率分别为(63.6±8.5)%,(69.0±11.7)%,(86.1 ±3.2)%。而未转染组和阴性对照组对CD70mRNA表达均无统计学差异。(4)siRNA3转染DCs,转染siRNA3组CD70mRNA明显低于对照组,CD70蛋白表达也明显低于对照组,未转染组和阴性对照组CD70蛋白的表达无明显差异。(5)PHA可以明显促进ITP患者DCs刺激CD4+T细胞增殖,ITP患者DCs转染siRNA3组的增殖低于阴性对照组,正常人DCs转染siRNA3组相对于转染阴性对照组无明显差异。(6)ITP患者和正常人转染siRNA3组分泌IFN-y均低于转染阴性对照组,分泌IL-10均高于阴性对照组。(7)在TGF-β诱导下,ITP患者和正常DCs转染siRNA3诱导正常来源CD4+T细胞分化的Treg细胞均低于转染阴性对照组。[结论]ITP患者的DCs比正常对照组更密集,可能是ITP患者中DCs相对于正常人功能亢进。siRNA可特异抑制树突细胞CD70基因的转录和表达。用siRNA干扰DC细胞CD70后抑制了 CD4+T细胞增殖,诱导分化Treg细胞功能减弱,逆转了 ITP患者Th1/Th2的极化状态。本研究结果为自身免疫疾病提供了新思路和治疗途径。[目的]原发性免疫血小板减少症(ITP)是一种自身免疫性疾病,其主要表现为血小板减少和出血风险增加。尽管CD83是成熟树突状细胞(DCs)的选择性标记物,但也表达于多种其他细胞,包括胸腺上皮细胞,T细胞,特别是调节性T细胞和B细胞。CD83和sCD83可能在自身免疫中发挥重要作用。在我们的研究中,我们研究ITP患者CD4+T细胞上的表达,以及经典的CD3/CD28信号和转化生长因子(TGF)-β刺激对CD4+T细胞CD83表达的影响以及它们在ITP中分化成CD4+CD25+FoxP3+诱导的调节性T(iTreg)细胞的研究。旨在探讨ITP患者CD4+T细胞亚群上CD83表达的情况以及与ITP患者病情和预后的相关性。[方法](1)76例ITP患者入组,均符合2009年国际专家共识的诊断标准,排除其他自身免疫病。(2)通过流式细胞仪检测ITP患者和正常人CD83+CD4+T呈现时间依赖性关系;不同分组的ITP患者与正常对照组检测各组细胞群的CD83+CD4+T细胞的表达情况以及与性别,年龄,病程,血小板计数等临床指标之间的相关性分析;(3)ITP患者和健康人外周血单个核细胞(PBMCs)体外培养,培养体系中分别加入 anti-CD3/28;anti-CD3/28+TGF-β;anti-CD3/28+TGF-β +CD83单克隆抗体,通过实时定量聚合酶链反应检测PBMCs中CD83的mRNA表达水平,流式细胞仪检测CD83的表达与CD4+CD25-、CD4+CD25+、CD4+CD25+FOXp3+Treg的关系。(4)酶联免疫吸附测定血浆中sCD83浓度,sCD83与血小板数的相关性分析。[结果]1.不同分组间CD83+CD4+T细胞表现为ITP患者初治组、活动期、缓解组和正常对照组分别为(6.47%±0.55%);(3.93%±0.46%);(2.97%±0.26%);(2.60%±0.33),初治组未用药组和活动期显著高于缓解组和正常对照组,差异均有统计学意义(P均0.01)。2.ITP患者和正常人外周血单个核细胞在抗CD3/CD28刺激下,CD83+CD4+T细胞表现出时间依赖性,ITP患者组在0天时CD83+CD4+T表达很低,而在第一天时CD83+CD4+T阳性的百分比开始上调(2.49±0.31%),第2天进一步显著增加(5.48±0.48%),到第三天开始降低至(1.97±0.32%)。3.在ITP患者组和正常人组的CD4+CD25-细胞CD83表达均较低,CD4+CD25+细胞CD83表达较高,CD4+CD25+FOXp3+Treg细胞CD83表达最高4.antiCD83下调CD83的表达,TGF-β上调CD83的表达。ITP患者组CD83分子mRNA的相对表达量显著高于正常对照组。ITP患者外周血浆中sCD83水平升高,与CD83+CD4+T表达相一致,并与血小板数目呈负相关(r=-0.438,p=0.007),而与性别和年龄无相关性。[结论]CD83+CD4+T细胞与ITP患者病程呈显著负相关。血小板计数与血浆sCD83水平呈显著负相关。随着病情的缓解,CD83+CD4+T细胞表达和血浆sCD83水平明显下调。CD83在Treg上表达升高可能与其免疫抑制功能异常有关,同时sCD83的水平未来可能可用来监测ITP患者病情变化。
[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.
【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R558.2
本文编号：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.
【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R558.2
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