课题一 维生素D受体及其基因多态性在再生障碍性贫血发病机制中的作用 课题二 IL-35在获得性再生障碍性贫血中的表达及其
发布时间：2018-08-14 13:53
【摘要】：背景：获得性再生障碍性贫血(aplastic anemia, AA)为一类以全血细胞减少和骨髓造血衰竭为特征的自身免疫性疾病。免疫介导的造血抑制是AA的主要发病机制。1 a,25-二羟维生素D3 [1a,25-Dihydroxyvitamin D3,1,25(OH)2D3]为维生素D的活性形式,除了调节机体钙磷代谢外,近期研究发现其与维生素D受体(vitamin D receptor, VDR)结合后尚可发挥免疫调节效应。已有研究证实1,25(OH)2D3和VDR在多种自身免疫性疾病发生发展中发挥关键作用,但其在AA发病机制中的作用尚未明确。目的：本研究旨在检测1,25(OH)2D3和VDR在AA患者中的表达,同时评价1,25(OH)2D3对AA患者外周血单个核细胞(peripheral blood mononuclear cells, PBMCs)的作用,探讨其在AA发病机制中的作用,为AA的免疫抑制治疗提供新的思路。方法：(1)通过ELISA方法检测血浆中25(OH)D3的表达,并进一步分析25(OH)D3水平与AA患者各临床参数之间的关系；(2)通过实时定量PCR的方法检测PBMCs以及1,25(OH)2D3处理后的PBMCs中VDR mRNA的表达水平；(3)通过CCK-8的方法检测1,25(OH)2D3对PBMCs增殖的影响；(4)将患者PBMCs用1,25(OH)2D3处理,未处理组作为对照。48小时后,采用ELISA的方法检测培养上清中TNF-α IFN-γ IL-17A、IL-10和TGF-β1的水平变化,实时定量PCR的方法检测T细胞相关转录因子T-bet. GATA3. RORyt和Foxp3 mRNA的表达变化,同时采用流式细胞术的方法检测1,25(OH)2D3处理的PBMCs内Thl (CD3+CD8-IFNy+)、Th2 (CD3+CD8-IL4+)、Tcl (CD3+CD8+IFNγ+)、Tc2 (CD3+CD8+IL4+)和Th17 (CD3+CD8-IL17+)细胞的比例变化。结果：(1)初治AA患者血浆25(OH)D3的水平与完全缓解患者、正常对照组无显著差异；(2)AA患者血浆25(OH)D3的水平与血小板计数、自然杀伤T细胞(natural killer T cells, NKT)比例呈显著正相关,与B细胞比例呈负相关；(3)初治AA患者PBMCs VDR mRNA表达水平显著低于正常对照组；(4)体外刺激实验显示1,25(OH)2D3抑制AA患者PBMCs的增殖;(5)外源性1,25(OH)2D3的加入明显抑制AA患者PBMCs分泌TNF-α、IFN-γ和IL-17A,并促进TGF-β1的产生；(6)1,25(OH)2D3抑制Th1、Tc1和Th17细胞的极化,同时促进Th2和Tc2细胞的极化,实时定量PCR的结果进一步证实1,25(OH)2D3抑制AA患者PBMCs内T-be、RORyt mRNA的表达,同时促进GATA3、Foxp3 mRNA的表达；(7)1,25(OH)2D3处理后正常对照组PBMCs中VDR mRNA的表达水平明显上调,AA患者却无明显变化。结论：初治AA患者PBMCs中VDR表达水平明显降低,低表达的VDR可能使维生素D-VDR通路信号传导减弱,从而导致AA的“高免疫”状态。适量的补充维生素D可通过增强信号传导来部分纠正AA免疫失耐受,有望成为一种新的辅助性治疗策略。背景：再生障碍性贫血(aplastic anemia, AA)是一种少见的可严重危及患者生命的骨髓衰竭性疾病,目前对其发病机制的研究主要集中在免疫调节紊乱、造血微环境缺陷、造血干/祖细胞数量和(或)功能缺陷及遗传易感性等方面。维生素D具有重要的免疫调节作用,其效应的发挥依赖于与维生素D受体(vitamin D receptor, VDR)的结合。VDR基因位于第12号染色体,包含多个单核苷酸多态性(SNP)位点。研究发现,VDR基因及其多态性与多种自身免疫性疾病的易感性相关。目前关于VDR基因多态性是否与AA发病相关尚未见报道。目的：本研究旨在探讨VDR基因四个多态性位点(rs2228570、rs1544410、rs7975232和rs731236)与AA易感性的关系,并评价VDR基因多态性与AA严重程度、治疗疗效及晚期克隆演变等临床特征的相关性。方法：采集AA患者和正常对照的外周血并提取DNA,采用聚合酶链反应-连接酶检测反应(PCR-LDR)的方法检测197例AA患者和135例健康对照rs1544410 (c.1024+283GA)、rs7975232 (c.1025-49GT)和rs731236(c.1056TC)的基因型。采用聚合酶链反应-限制性片段长度多态性(PCR-RFLP)方法检测rs2228570(c.2TC)的基因型。应用SPSS 17.0软件统计分析VDR基因多态性位点与AA易感性、疾病严重程度、治疗疗效、克隆演变及其他临床特征的相关性。结果：(1)VDR基因四个多态性位点的基因型均符合Hardy-Weinberg平衡。(2)VDR基因rs1544410、rs7975232和rs731236三个位点之间存在显著的遗传连锁不平衡。 (3)在VDR基因的四个多态性位点中,AA患者rs1544410位点GG基因型和G等位基因频率显著高于正常对照组,而其他三个多态性位点与AA易感性无关。 (4)进一步分析发现rs1544410和rs7975232位点与非重型AA的发病相关,而rs2228570位点与重型AA的发病相关。 (5)AA患者rs2228570位点的基因型与治疗疗效相关：CC及CT基因型患者预后好于TT基因型者。(6)AA患者rs2228570位点的基因型与晚期克隆演变亦密切相关：TT基因型携带者更易于转化为骨髓增生异常综合征／急性髓系白血病,而CT基因型患者更易进展为阵发性睡眠性血红蛋白尿症。结论：我们的研究结果表明,在中国人群中,VDR基因多态性与AA的易感性、疾病严重程度及预后密切相关。背景：获得性再生障碍性贫血(aplastic anemia, AA)是一种自身反应性T细胞攻击靶器官骨髓所致的骨髓衰竭性疾病。IL-35为近年新发现的一种调节性细胞因子,是IL-12家族的新成员,主要由调节性T细胞(Tregs)分泌产生。目前认为IL-35为Tregs发挥免疫抑制作用的主要效应分子。此外,IL-35尚可诱导T细胞分化为可分泌IL-35的诱导型调节性T细胞(iTR35),进一步维持机体的免疫耐受状态。越来越多的证据表明IL-35在调控机体免疫稳态中发挥关键作用,但其在AA中的表达情况以及是否参与AA的发病机制尚未明确。目的：本研究旨在检测AA患者血浆中IL-35的表达情况,并评估IL-35对T细胞免疫反应的作用,探讨其在AA发病机制中的作用,为基于IL-35的靶向治疗提供理论依据。方法： (1)收集患者临床资料；(2)通过ELISA方法检测血浆中IL-35的表达,并进一步分析IL-35水平与AA患者各临床参数之间的关系; (3)通过实时定量PCR的方法检测外周血单个核细胞(PBMCs)中IL-35两个亚基p35和EBI3 mRNA的表达水平； (4)通过Brdu掺入法检测IL-35对PBMCs增殖的影响；(5)将患者和正常对照PBMCs用IL-35处理,未处理组作为对照。48小时后,采用ELISA的方法检测培养上清中TNF-α IFN-γ、IL-17A. IL-10和TGF-β1的水平变化,流式细胞术和细胞计数相结合的方法检测CD4+/CD8+T细胞、Thl (CD3+CD8-IFNy+)、Th2 ( CD3+CD8-IL4+)、Tcl (CD3+CD8+IFNy+)、Tc2 (CD3+CD8+IL4+)和Th17 ( CD3+CD8-IL17+)细胞的比例和绝对数变化,同时采用实时定量PCR的方法检测T细胞相关转录因子T-bet、GATA3、RORγt和Foxp3 mRNA的表达变化。结果：(1)通过ELISA检测,我们发现初治AA患者血浆中IL-35的水平明显低于完全缓解(CR)患者和正常对照组,而CR患者和正常对照组之间无显著差异,进一步分析发现IL-35水平与疾病的严重程度密切相关。(2)在初治AA患者,血浆IL-35的水平与血小板计数、中性粒细胞绝对值和网织红细胞绝对值呈显著正相关,与淋巴细胞比例呈负相关。(3)实时定量PCR的结果显示,初治重型AA患者p35和EBI3 mRNA表达水平低于正常对照组,而初治非重型AA与正常对照组之间无显著差异。(4)体外刺激实验显示IL-35能抑制AA患者PBMCs的增殖,进而行细胞计数和流式细胞术发现IL-35能抑制CD4+和CD8+T细胞的增殖。(5)外源性IL-35的加入明显抑制AA患者PBMCs分泌TNF-α IFN-γ和IL-17A,同时促进TGF-β的产生。(6)IL-35抑制Th1、Tcl和Th17细胞的极化,同时促进Th2和Tc2细胞的极化,实时定量PCR的结果证实IL-35抑制AA患者PBMCs内T-bet和RORyt mRNA的表达,同时促进GATA3 mRNA的表达,对Foxp3 mRNA的表达无明显影响。结论：初治AA患者体内IL-35的表达水平降低,且与疾病严重程度密切相关,IL-35低表达可能致使Tregs失能,进而导致AA的免疫失耐受状态,提示IL-35在AA的发病机制中发挥关键作用。
[Abstract]:BACKGROUND: Acquired aplastic anemia (AA) is an autoimmune disease characterized by pancytopenia and bone marrow hematopoietic failure. Immune-mediated hematopoietic suppression is the main pathogenesis of AA. In addition to the regulation of calcium and phosphorus metabolism, recent studies have found that the binding of vitamin D receptor (VDR) with 1,25 (OH) 2D3 and VDR may play an important role in the pathogenesis of AA. Objective To detect the expression of 1,25 (OH) 2D3 and VDR in patients with AA, and to evaluate the effect of 1,25 (OH) 2D3 on peripheral blood mononuclear cells (PBMCs) in patients with AA, and to explore the role of 1,25 (OH) 2D3 in the pathogenesis of AA. The expression of VDR mRNA in PBMCs and PBMCs treated with 1,25 (OH) 2D3 was detected by real-time quantitative PCR; (3) The effect of 1,25 (OH) 2D3 on the proliferation of PBMCs was detected by CCK-8; (4) PBMCs were treated with 1,25 (OH) 2D3. After 48 hours, the levels of TNF-alpha IFN-gamma IL-17A, IL-10 and TGF-beta 1 in culture supernatant were detected by ELISA, and the expression of T-cell-related transcription factors T-bet.GATA3.RORyt and Foxp3 mRNA were detected by real-time quantitative PCR, and the expression of PBMC treated with 1,25(OH)2D3 was detected by flow cytometry. The proportion of Thl (CD3 + CD8 - IFNy +), Th2 (CD3 + CD8 - IL4 +), Tcl (CD3 + CD8 + IFN gamma +), Tc2 (CD3 + CD8 + IL4 +) and Th17 (CD3 + CD8 - IL17 +) cells changed within s. Results: (1) There was no significant difference in plasma 25 (OH) D3 levels and platelet counts, and natural killer T (NK) between AA patients and complete remission patients. The proportion of natural killer T cells (NKT) was significantly positively correlated with the proportion of B cells, and negatively correlated with the proportion of B cells; (3) The expression level of VDR mRNA in PBMCs of newly treated AA patients was significantly lower than that of normal controls; (4) In vitro stimulation test showed that 1,25 (OH) 2D3 inhibited the proliferation of PBMCs of AA patients; (5) Exogenous 1,25 (OH) 2D3 significantly inhibited the secretion of TNF-2 by PBMCs of AA patients. Alpha, IFN-gamma and IL-17A, and promote the production of TGF-beta 1; (6) 1,25 (OH) 2D3 inhibits the polarization of Th1, Tc 1 and Th17 cells, and promotes the polarization of Th2 and Tc2 cells. Real-time quantitative PCR further confirmed that 1,25 (OH) 2D3 inhibits the expression of T-be and RORyt mRNA in PBMCs of AA patients, and promotes the expression of GATA3 and Foxp3 mRNA; (7) 1,25 (OH) 2D3 treatment is positive. Conclusion: The expression of VDR in PBMCs of newly treated AA patients is significantly lower than that of control group, and the low expression of VDR may weaken the signal transduction of vitamin D-VDR pathway and lead to the "high immunity" state of AA. BACKGROUND: Aplastic anemia (AA) is a rare bone marrow failure disease that can seriously endanger the life of patients. Currently, studies on its pathogenesis mainly focus on immunomodulatory disorders, hematopoietic microenvironment defects, hematopoietic stem/progenitor. Vitamin D plays an important role in immune regulation. Its effect depends on binding to vitamin D receptor (VDR). VDR gene is located on chromosome 12 and contains multiple single nucleotide polymorphisms (SNP) loci. Objective: To investigate the relationship between the susceptibility to AA and the polymorphism of VDR gene (rs2228570, rs1544410, rs7975232 and rs731236), and to evaluate the relationship between the polymorphism of VDR gene and the severity and treatment of AA. Methods: DNA was extracted from peripheral blood of AA patients and normal controls, and the bases of rs1544410 (c. 1024 + 283GA), rs7975232 (c. 1025-49GT) and rs731236 (c. 1056TC) were detected by polymerase chain reaction-ligase assay (PCR-LDR) in 197 AA patients and 135 healthy controls. Genotype. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used to detect the genotype of rs2228570 (c.2TC). The correlation between VDR gene polymorphism and AA susceptibility, disease severity, therapeutic efficacy, clonal evolution and other clinical features was analyzed by SPSS 17.0 software. (2) There was a significant genetic linkage imbalance among the three VDR loci, rs1544410, rs797523 2 and rs731236. (3) Among the four polymorphic loci of VDR gene, the frequency of GG genotype and G allele at rs1544410 locus in AA patients was significantly higher than that in normal controls, while the other three polymorphisms were higher. Further analysis revealed that rs1544410 and rs7975232 loci were associated with non-severe AA, while rs2228570 loci were associated with severe AA. (5) The genotype of rs2228570 locus in AA patients was associated with therapeutic efficacy: the prognosis of CC and CT genotypes was better than that of TT genotypes. (6) The rs2228570 locus in AA patients was associated with severe AA. Genotypes are also closely related to late clonal evolution: TT carriers are more likely to develop myelodysplastic syndrome/acute myeloid leukemia, while CT carriers are more likely to develop paroxysmal nocturnal hemoglobinuria. BACKGROUND: Acquired aplastic anemia (AA) is a bone marrow failure disease caused by autoreactive T cells attacking bone marrow of target organs. IL-35 is a newly discovered regulatory cytokine, a new member of the IL-12 family, mainly composed of regulatory T cells (Tregs). In addition, IL-35 can induce T cells to differentiate into inducible regulatory T cells (iTR35) secreting IL-35, which can further maintain the immune tolerance of the body. Objective: To detect the expression of IL-35 in plasma of AA patients and evaluate the effect of IL-35 on T cell immune response, and to explore its role in the pathogenesis of AA, so as to provide theoretical basis for targeted therapy based on IL-35. (3) The expression of IL-35 and EBI3 mRNA in peripheral blood mononuclear cells (PBMCs) was detected by real-time quantitative PCR; (4) The expression of IL-35 and EBI3 mRNA in PBMCs was detected by Brdu incorporation method; (4) The expression of IL-35 was detected by Brdu incorporation method. After 48 hours, the levels of TNF-alpha IFN-gamma, IL-17A.IL-10 and TGF-beta 1 in culture supernatant were detected by ELISA, and CD4+/CD8+T cells, Thl (CD3+CD8-IFNy+), Th2 (Th2+CD8-IFNy+) were detected by flow cytometry and cell counting. (CD3+CD8-IL4+), Tcl (CD3+CD8+IFNy+), Tc2 (CD3+CD8+IL4+) and Th17 (CD3+CD8-IL17+) cell ratios and absolute number changes, while real-time quantitative PCR method was used to detect T-bet, GATA3, ROR gamma T and Foxp3 mRNA expression changes. Results: (1) Through ELISA detection, we found that the plasma of newly treated AA patients with IL-35. The level of IL-35 was significantly lower than that of CR patients and normal control group, but there was no significant difference between CR patients and normal control group. Further analysis showed that the level of IL-35 was closely related to the severity of the disease. (2) In newly treated AA patients, the levels of IL-35 were significantly correlated with platelet count, neutrophil absolute value and reticulocyte absolute value. (3) Real-time quantitative PCR showed that the expression of p35 and EBI3 mRNA in newly treated severe AA patients was lower than that in the normal control group, but there was no significant difference between the newly treated non-severe AA patients and the normal control group. (4) In vitro stimulation test showed that IL-35 could inhibit the proliferation of PBMCs in AA patients, and then the cell count and flow. IL-35 could inhibit the proliferation of CD4+ and CD8+ T cells. (5) Exogenous IL-35 significantly inhibited the secretion of TNF-alpha IFN-gamma and IL-17A by PBMCs in AA patients, and promoted the production of TGF-beta. (6) IL-35 inhibited the polarization of Th1, Tcl and Th17 cells, and promoted the polarization of Th2 and Tc2 cells. Real-time quantitative PCR results confirmed that IL-35 inhibited the production of TNF-alpha IFN-gamma and IL-17A by PBMCs in AA patients. The expression of T-bet and RORyt mRNA in BMCs and GATA3 mRNA had no significant effect on the expression of Foxp3 mRNA. Conclusion: The expression of IL-35 in newly treated AA patients was decreased, which was closely related to the severity of the disease. The low expression of IL-35 may cause Tregs disability, which may lead to the immune intolerance of AA, suggesting that IL-35 may occur in AA. Play a key role in the disease mechanism.
【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】：2016
【分类号】：R556.5
本文编号：2183068
[Abstract]:BACKGROUND: Acquired aplastic anemia (AA) is an autoimmune disease characterized by pancytopenia and bone marrow hematopoietic failure. Immune-mediated hematopoietic suppression is the main pathogenesis of AA. In addition to the regulation of calcium and phosphorus metabolism, recent studies have found that the binding of vitamin D receptor (VDR) with 1,25 (OH) 2D3 and VDR may play an important role in the pathogenesis of AA. Objective To detect the expression of 1,25 (OH) 2D3 and VDR in patients with AA, and to evaluate the effect of 1,25 (OH) 2D3 on peripheral blood mononuclear cells (PBMCs) in patients with AA, and to explore the role of 1,25 (OH) 2D3 in the pathogenesis of AA. The expression of VDR mRNA in PBMCs and PBMCs treated with 1,25 (OH) 2D3 was detected by real-time quantitative PCR; (3) The effect of 1,25 (OH) 2D3 on the proliferation of PBMCs was detected by CCK-8; (4) PBMCs were treated with 1,25 (OH) 2D3. After 48 hours, the levels of TNF-alpha IFN-gamma IL-17A, IL-10 and TGF-beta 1 in culture supernatant were detected by ELISA, and the expression of T-cell-related transcription factors T-bet.GATA3.RORyt and Foxp3 mRNA were detected by real-time quantitative PCR, and the expression of PBMC treated with 1,25(OH)2D3 was detected by flow cytometry. The proportion of Thl (CD3 + CD8 - IFNy +), Th2 (CD3 + CD8 - IL4 +), Tcl (CD3 + CD8 + IFN gamma +), Tc2 (CD3 + CD8 + IL4 +) and Th17 (CD3 + CD8 - IL17 +) cells changed within s. Results: (1) There was no significant difference in plasma 25 (OH) D3 levels and platelet counts, and natural killer T (NK) between AA patients and complete remission patients. The proportion of natural killer T cells (NKT) was significantly positively correlated with the proportion of B cells, and negatively correlated with the proportion of B cells; (3) The expression level of VDR mRNA in PBMCs of newly treated AA patients was significantly lower than that of normal controls; (4) In vitro stimulation test showed that 1,25 (OH) 2D3 inhibited the proliferation of PBMCs of AA patients; (5) Exogenous 1,25 (OH) 2D3 significantly inhibited the secretion of TNF-2 by PBMCs of AA patients. Alpha, IFN-gamma and IL-17A, and promote the production of TGF-beta 1; (6) 1,25 (OH) 2D3 inhibits the polarization of Th1, Tc 1 and Th17 cells, and promotes the polarization of Th2 and Tc2 cells. Real-time quantitative PCR further confirmed that 1,25 (OH) 2D3 inhibits the expression of T-be and RORyt mRNA in PBMCs of AA patients, and promotes the expression of GATA3 and Foxp3 mRNA; (7) 1,25 (OH) 2D3 treatment is positive. Conclusion: The expression of VDR in PBMCs of newly treated AA patients is significantly lower than that of control group, and the low expression of VDR may weaken the signal transduction of vitamin D-VDR pathway and lead to the "high immunity" state of AA. BACKGROUND: Aplastic anemia (AA) is a rare bone marrow failure disease that can seriously endanger the life of patients. Currently, studies on its pathogenesis mainly focus on immunomodulatory disorders, hematopoietic microenvironment defects, hematopoietic stem/progenitor. Vitamin D plays an important role in immune regulation. Its effect depends on binding to vitamin D receptor (VDR). VDR gene is located on chromosome 12 and contains multiple single nucleotide polymorphisms (SNP) loci. Objective: To investigate the relationship between the susceptibility to AA and the polymorphism of VDR gene (rs2228570, rs1544410, rs7975232 and rs731236), and to evaluate the relationship between the polymorphism of VDR gene and the severity and treatment of AA. Methods: DNA was extracted from peripheral blood of AA patients and normal controls, and the bases of rs1544410 (c. 1024 + 283GA), rs7975232 (c. 1025-49GT) and rs731236 (c. 1056TC) were detected by polymerase chain reaction-ligase assay (PCR-LDR) in 197 AA patients and 135 healthy controls. Genotype. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used to detect the genotype of rs2228570 (c.2TC). The correlation between VDR gene polymorphism and AA susceptibility, disease severity, therapeutic efficacy, clonal evolution and other clinical features was analyzed by SPSS 17.0 software. (2) There was a significant genetic linkage imbalance among the three VDR loci, rs1544410, rs797523 2 and rs731236. (3) Among the four polymorphic loci of VDR gene, the frequency of GG genotype and G allele at rs1544410 locus in AA patients was significantly higher than that in normal controls, while the other three polymorphisms were higher. Further analysis revealed that rs1544410 and rs7975232 loci were associated with non-severe AA, while rs2228570 loci were associated with severe AA. (5) The genotype of rs2228570 locus in AA patients was associated with therapeutic efficacy: the prognosis of CC and CT genotypes was better than that of TT genotypes. (6) The rs2228570 locus in AA patients was associated with severe AA. Genotypes are also closely related to late clonal evolution: TT carriers are more likely to develop myelodysplastic syndrome/acute myeloid leukemia, while CT carriers are more likely to develop paroxysmal nocturnal hemoglobinuria. BACKGROUND: Acquired aplastic anemia (AA) is a bone marrow failure disease caused by autoreactive T cells attacking bone marrow of target organs. IL-35 is a newly discovered regulatory cytokine, a new member of the IL-12 family, mainly composed of regulatory T cells (Tregs). In addition, IL-35 can induce T cells to differentiate into inducible regulatory T cells (iTR35) secreting IL-35, which can further maintain the immune tolerance of the body. Objective: To detect the expression of IL-35 in plasma of AA patients and evaluate the effect of IL-35 on T cell immune response, and to explore its role in the pathogenesis of AA, so as to provide theoretical basis for targeted therapy based on IL-35. (3) The expression of IL-35 and EBI3 mRNA in peripheral blood mononuclear cells (PBMCs) was detected by real-time quantitative PCR; (4) The expression of IL-35 and EBI3 mRNA in PBMCs was detected by Brdu incorporation method; (4) The expression of IL-35 was detected by Brdu incorporation method. After 48 hours, the levels of TNF-alpha IFN-gamma, IL-17A.IL-10 and TGF-beta 1 in culture supernatant were detected by ELISA, and CD4+/CD8+T cells, Thl (CD3+CD8-IFNy+), Th2 (Th2+CD8-IFNy+) were detected by flow cytometry and cell counting. (CD3+CD8-IL4+), Tcl (CD3+CD8+IFNy+), Tc2 (CD3+CD8+IL4+) and Th17 (CD3+CD8-IL17+) cell ratios and absolute number changes, while real-time quantitative PCR method was used to detect T-bet, GATA3, ROR gamma T and Foxp3 mRNA expression changes. Results: (1) Through ELISA detection, we found that the plasma of newly treated AA patients with IL-35. The level of IL-35 was significantly lower than that of CR patients and normal control group, but there was no significant difference between CR patients and normal control group. Further analysis showed that the level of IL-35 was closely related to the severity of the disease. (2) In newly treated AA patients, the levels of IL-35 were significantly correlated with platelet count, neutrophil absolute value and reticulocyte absolute value. (3) Real-time quantitative PCR showed that the expression of p35 and EBI3 mRNA in newly treated severe AA patients was lower than that in the normal control group, but there was no significant difference between the newly treated non-severe AA patients and the normal control group. (4) In vitro stimulation test showed that IL-35 could inhibit the proliferation of PBMCs in AA patients, and then the cell count and flow. IL-35 could inhibit the proliferation of CD4+ and CD8+ T cells. (5) Exogenous IL-35 significantly inhibited the secretion of TNF-alpha IFN-gamma and IL-17A by PBMCs in AA patients, and promoted the production of TGF-beta. (6) IL-35 inhibited the polarization of Th1, Tcl and Th17 cells, and promoted the polarization of Th2 and Tc2 cells. Real-time quantitative PCR results confirmed that IL-35 inhibited the production of TNF-alpha IFN-gamma and IL-17A by PBMCs in AA patients. The expression of T-bet and RORyt mRNA in BMCs and GATA3 mRNA had no significant effect on the expression of Foxp3 mRNA. Conclusion: The expression of IL-35 in newly treated AA patients was decreased, which was closely related to the severity of the disease. The low expression of IL-35 may cause Tregs disability, which may lead to the immune intolerance of AA, suggesting that IL-35 may occur in AA. Play a key role in the disease mechanism.
【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】：2016
【分类号】：R556.5
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