从DC-Th轴探讨CD86-siRNA基因修饰树突状细胞在变应性鼻炎中的作用研究

发布时间：2018-06-05 14:06

本文选题：变应性鼻炎 + siRNA　；参考：《重庆医科大学》2015年博士论文

【摘要】：目的：变应性鼻炎(allergic rhinitis, AR)是发生在鼻粘膜的变态反应性疾病,其发病以儿童和青壮年居多。据WHO近年公布的数据表明全世界目前超过5亿人罹患此病。流行病学资料也显示,在2008年美国变应性鼻炎的发病率高达16.7%,在慢性病中居第5位,而AR也是儿童最常见的慢性疾病,每年用于诊治变应性鼻炎的费用高达数十亿美元。而在我国,2007年公布的国内11个中心城市的流行病学资料,成人自报患病率介于9-24.6%,儿童则介于1.8-13.67%。变应性鼻炎虽不会引起严重后果,但能很大程度影响患者日常生活质量。目前,对变应性鼻炎最常用的治疗方案是鼻喷糖皮质激素和口服抗组胺药,该方法虽然能快速控制鼻过敏性炎症,有效缓解临床症状,却不能阻止变应性鼻炎反复发作,距治愈的目标尚远。研究表明,仅有约60%的患者对变应性鼻炎的治疗总体上感到非常满意,且随着时间的推移,药物治疗的疗效会逐渐减弱,这些治疗上的困难究其原因在于变应性鼻炎的发病机制尚未完全明了,难以探索出更为有效的治疗手段。因此,加大对变应性鼻炎发病机制的研究和防治措施的探讨是目前耳鼻咽喉科学领域高度重视和急待解决的重点之一。变应性鼻炎的病因尚不明确,一般认为与特应性个体、遗传学背景及环境因素有关。大量的实验研究证实,鼻变态反应的发病机制是以Th2反应为主的变态反应性疾病,即’Th1/Th2失衡引发的下游生物效应导致变应性鼻炎的发生,但最近随着研究的进一步深入,有些学者提出新识别的T细胞亚群可能对Th2细胞的分化有调节作用,如CD4+CD25+调节性T细胞对Th2细胞所介导的呼吸道变应性炎症发挥强大的抑制作用。变应性个体可能存在Treg/Th2失衡,但其复杂的信号传导途径和机制仍在研究和探索中,这必将为变应性鼻炎提供新的治疗靶点。DC是体内功能最强大的专职抗原提呈细胞,也是唯一能使初始T淋巴细胞活化的APC,在免疫系统中处于启动、调控并维持免疫应答的中心环节。有研究证实变应性鼻炎体内Th2的极化状态与DC细胞的功能异常有关,DC成为研究影响T细胞活化与增殖的重要靶细胞。未成熟DC由于其表面缺乏共刺激因子,在体内或体外均显示出诱导Treg细胞的特性,因此采用基因修饰的方法对DC加以修饰,使其保持稳定的未成熟DC的特性,从而达到抑制效应T细胞的活化而增强Treg细胞活化的目的。本课题拟以AR为研究对象,阐明AR中是否存在Treg/Th2失衡并以DC-Th细胞间相互作用为主线,以慢病毒载体CD86-siRNA对DC的调控为切入点,阐述AR中基因修饰DC对Treg/Th2失衡的调控,说明Th失衡的上游事件及其免疫调控因素,为AR发病机制的研究提供新的思路。方法：1、纳入排除标准：本研究严格按照中华医学会变应性鼻炎诊断和治疗指南(2009,武夷山),所有入组患者均来自重庆医科大学附属儿童医院耳鼻咽喉科门诊,均为首次诊断,未接受变应性鼻炎相关治疗,对照组排除有感染性的、占位性的、药物诱导性鼻炎和有任何并发症的患者,在获得其家长知情同意后,将其纳入研究对象。2、探讨AR患者与正常对照外周血中Treg/Th2细胞亚群分布及与临床症状的关系、Treg/Th2相关细胞因子及转录因子的表达差异。(1)按照ELISA试剂盒操作步骤检测AR患者与正常对照外周血中总IgE、IL-4、IL-5、TGF-β1的含量。(2) RT-PCR扩增检测外周血转录因子FOXP、GATA-3mRNA表达情况。(3)采用Western Blot方法检测AR患者及正常对照组外周血PBMC中FOXP3、GATA-3蛋白水平。3、慢病毒载体siRNA序列转染人树突状细胞沉默CD86基因对Treg/Th2细胞分化的影响(1)慢病毒载体siRNA的构建(2) MACS分选人外周血CD14+单核细胞并诱导成熟DC(3) MACS分选人外周血CD4十T细胞(4)携带特异性siRNA的慢病毒转染DC(5)AR组未转染DC、转染后DC、正常对照组DC与外周血CD4+T细胞共培养将未转染DC、转染后DC与外周血CD4+T细胞共培养,按照ELISA试剂盒操作步骤检测共培养上清液中IL-4、IL-5、TGF-β1的含量,RT-PCR法检测FOXP3、GATA-3mRNA水平,Western Blotting方法检测FOXP3、GATA-3蛋白水平,以评价CD86基因修饰后DC对Th细胞分化的影响。结果：(1)AR组与对照组相比,血清总IgE水平、嗜酸性粒细胞比例、T5SS均明显升高,且血清总IgE水平、嗜酸性粒细胞比例与T5SS呈正相关,而TGF-β1/IL-4、TGF-β1/IL-5、FOXP3 mRNA/GATA3 mRNA、FOXP3 protein/GATA3 protein比值与T5SS呈负相关。(2) ELISA检测AR血清中IL-4, IL-5, TGF-β1的表达,AR组与对照组相比,IL-4,IL-5明显增高,而TGF-β1的水平AR组明显低于对照组。RT-PCR检测Treg转录因子FOXP3 mRNA的表达,AR组明显低于对照组,而AR组Th2转录因子GATA3 mRNA的表达明显高于对照组。Western blotting检测FOXP3与GATA3蛋白表达水平,结果GATA3, FOXP3蛋白表达趋势与二者mRNA的表达趋势一致。(3)应用MACS法从血液中成功提取CD14+单核细胞并刺激转化成熟DC,成功提取CD4+T淋巴细胞,建立共培养体系(DC/CD4+T1:4);应用CD86-siRNA慢病毒成功转染DC,MOI值20,转染率约为60.2%；转染后DC表面分子标志CD86的表达明显下降；AR组未转染,转染后DC及正常对照组DC分别与CD4+T共培养,上清液中Th2型细胞因子IL-4、IL-5在siRNA转染后其表达水平明显低于未转染组,但仍明显高于正常对照组,而Treg型细胞因子TGF-β1在AR未转染组的表达低于正常对照,siRNA转染后其表达明显高于未转染组,但仍低于正常对照组；Th2型转录因子GATA3 mRNA水平及蛋白表达水平siRNA转染后明显低于未转染组,但高于正常对照组,而Treg型转录因子FOXP3 mRNA水平及蛋白表达水平,AR组转染后表达高于未转染组,二组均低于正常对照组。结论：(1)AR患者体内FOXP3 mRNA和蛋白表达降低,而GATA3 mRNA和蛋白表达升高,FOXP3 mRNA/GATA3 mRNA、FOXP3蛋白/GATA3蛋白与AR的疾病严重程度(T5SS)呈负相关,提示AR患者体内可能存在另外一种免疫失衡即Treg/Th2失衡。(2)通过体外细胞共培养实验,应用携带特异性CD86-siRNA序列慢病毒载体转染人树突状细胞沉默CD86基因,结果显示树突状细胞表面分子CD86的表达明显减少,这种稳定的、未成熟DC影响了T细胞的分化,表现为Treg表达升高而Th2细胞表达降低,提示AR患者可以通过基因修饰DC来调节T细胞的活化,即改变Treg/Th2平衡,从而说明DC-Th轴在AR中的作用,为治疗AR提供新的思路。
[Abstract]:Objective: allergic rhinitis (AR) is a allergic rhinitis (AR) occurring in the nasal mucosa, which is mostly in children and young adults. According to data published in recent years, more than 500 million people worldwide are suffering from this disease. Epidemiological data also show that in 2008, the incidence of allergic rhinitis in the United States was as high as 16.7%, in chronic diseases. In the fifth place, AR is also the most common chronic disease of children. The cost of treating allergic rhinitis is as high as billions of dollars each year. In China, in China, the epidemiological data of the 11 central cities published in 2007, the adult self reported prevalence rate was between 9-24.6%, and children with 1.8-13.67%. allergic rhinitis did not cause serious consequences, But at present, the most commonly used treatment for allergic rhinitis is nasal spray glucocorticoid and oral antihistamine. Although it can quickly control allergic rhinitis and relieve clinical symptoms, it can not prevent allergic rhinitis from repeated attacks and far away from the target of cure. Only about 60% of the patients are very satisfied with the treatment of allergic rhinitis, and the effect of drug therapy will gradually weaken as time goes on. The reason for these difficulties is that the pathogenesis of allergic rhinitis is not completely clear, and it is difficult to explore more effective treatment. Therefore, increase the allergic reaction. The study and prevention of the pathogenesis of rhinitis is one of the most important and urgent problems in the field of Otorhinolaryngology at present. The etiology of allergic rhinitis is still not clear, and it is generally considered to be related to the individual, genetic background and environmental factors. A large number of experimental studies have confirmed that the pathogenesis of nasal allergy is Th2 The reaction mainly allergic disease, that is, the downstream biological effect caused by the Th1/Th2 imbalance leads to the occurrence of allergic rhinitis, but recently, with the further research, some newly identified T cell subsets may regulate the differentiation of Th2 cells, such as the respiration of the CD4+ CD25+ regulatory T cells to Th2 cells. Allergic inflammation plays a strong inhibitory role. Allergic individuals may have Treg/Th2 imbalance, but their complex signal transduction pathways and mechanisms are still being studied and explored. This will certainly provide a new therapeutic target for allergic rhinitis,.DC, the most powerful specific antigen presenting cell in the body, and the only one that can make the initial T lymphocyte. Activated APC, which is activated in the immune system, regulates and maintains the central link in the immune response. Studies have shown that the polarization of Th2 in allergic rhinitis is related to the dysfunction of DC cells. DC has become an important target cell for the study of the activation and proliferation of T cells. Immature DC is in the body or body on its surface. Both showed the characteristics of Treg cells, so DC was modified by gene modification to maintain the stability of the immature DC, thus achieving the purpose of inhibiting the activation of the T cells and enhancing the activation of Treg cells. This subject is to use AR as the research object to clarify whether there is a Treg/Th2 imbalance in AR and the DC-Th cell between the cells. The interaction is the main line. Taking the regulation of DC by the lentivirus carrier CD86-siRNA as the breakthrough point, this paper expounds the regulation of the Treg/Th2 imbalance in the gene modified DC in AR, explains the upstream events of the Th imbalance and the immune regulation factors, and provides a new way of thinking for the study of the pathogenesis of AR. Method: 1, the exclusion criteria are included: This study is strictly according to the Chinese Medical Association. The guidelines for the diagnosis and treatment of allergic rhinitis (2009, Wuyishan), all the patients from the Department of Otolaryngology, affiliated to the Affiliated Children's Hospital of Medical University Of Chongqing, were first diagnosed for the first time without allergic rhinitis related treatment. The control group excluded infectious, occupying, drug induced rhinitis and any patients with any complications. After parents' informed consent, they were included in the study.2. The distribution of Treg/Th2 cell subsets in AR patients and normal control peripheral blood and the relationship with the clinical symptoms, the difference in the expression of Treg/Th2 related cytokines and transcription factors. (1) to detect the total IgE, IL-4, IL-5, TGF- in the peripheral blood of AR patients and normal control peripheral blood according to the ELISA kit operation procedure. The content of beta 1. (2) RT-PCR amplification was used to detect the transcription factor FOXP and GATA-3mRNA expression in peripheral blood. (3) Western Blot was used to detect FOXP3, GATA-3 protein level.3 in the peripheral blood of AR patients and normal control group, and the effect of lentivirus carrier siRNA sequence on the differentiation of human dendritic cells (1) lentivirus carrier The construction of body siRNA (2) MACS selected peripheral blood CD14+ mononuclear cells and induced mature DC (3) MACS candidates in peripheral blood CD4 ten T cells (4) carrying specific siRNA in the lentivirus transfected DC (5) AR group without DC, after transfection, the normal control group and peripheral blood cells were co cultured with peripheral blood cells. The content of IL-4, IL-5, TGF- beta 1 in the co culture supernatant was detected according to the operation procedure of the ELISA kit. FOXP3, GATA-3mRNA level was detected by RT-PCR, Western Blotting method was used to detect FOXP3, GATA-3 protein level, and the effect of CD86 gene modification on differentiation of cells was evaluated. The proportion of granulocyte, T5SS, and serum total IgE level, eosinophil ratio was positively correlated with T5SS, while TGF- beta 1/IL-4, TGF- beta 1/IL-5, FOXP3 mRNA/GATA3 mRNA and FOXP3 protein/GATA3 ratio were negatively correlated. The level of TGF- beta 1 in the level AR group was significantly lower than that in the control group, and the expression of Treg transcription factor FOXP3 mRNA was significantly lower than that in the control group. The AR group was significantly lower than the control group, while the expression of GATA3 mRNA in AR group Th2 transcriptional factor was significantly higher than that of the control group. (3) (3) CD14+ mononuclear cells were successfully extracted from blood by MACS method and stimulated to transform mature DC, CD4+T lymphocytes were successfully extracted and co culture system (DC/CD4+T1:4) was successfully extracted; DC was successfully transfected with CD86-siRNA lentivirus, MOI value was 20, and the transfection rate was about 60.2%; the expression of CD86 of DC surface molecules after transfection was obviously decreased; AR after transfection; AR Group DC and normal control group DC were co cultured with CD4+T after transfection. The expression level of Th2 type cytokine in the supernatant was significantly lower than that in the normal control group, but the expression level of IL-5 in the supernatant was significantly lower than that in the normal control group, but the expression of Treg type cytokine TGF- beta 1 in the non transfected AR group was lower than that in the normal control group. The expression of the Treg type cytokine was lower than that of the normal control group. The expression of the Treg type cytokine was clearly expressed after the transfection. The level of GATA3 mRNA and protein expression level of Th2 type transcription factor siRNA were significantly lower than that of untransfected group, but higher than that of normal control group, but the level of FOXP3 mRNA and protein expression level of Treg type transcription factor were higher than that of non transfected group, and the two groups were all lower than those of normal control group. Conclusion: (1) the expression of FOXP3 mRNA and protein in AR patients decreased and the expression of GATA3 mRNA and protein increased, FOXP3 mRNA/GATA3 mRNA, FOXP3 protein /GATA3 protein was negatively correlated with AR disease severity (T5SS), suggesting that there may be another immune imbalance in the patient's body. (2) through in vitro cell co culture experiment, The expression of CD86 gene was silenced by human dendritic cells transfected with specific CD86-siRNA lentivirus vector. The results showed that the expression of CD86 in the surface of dendritic cells decreased obviously. This stable, immature DC affected the differentiation of T cells, which showed that the expression of Treg was increased and the expression of Th2 cells decreased, suggesting that AR patients could be modified by gene modification. DC regulates the activation of T cells, that is, changing the Treg/Th2 balance, thus explaining the role of DC-Th axis in AR, and providing new ideas for AR treatment.
【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R765.21

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