转录因子RORα对支气管哮喘的免疫调控作用

发布时间：2018-06-23 20:33

  本文选题：RORα + 支气管哮喘　； 参考：《江苏大学》2017年博士论文

【摘要】：背景支气管哮喘是一类由多种免疫细胞参与的慢性炎症性气道疾病,可发生于任何年龄。近年来,越来越多的研究发现,支气管哮喘患者体内存在着一群非T非B的免疫细胞,即固有淋巴细胞(innate lymphoid cells, ILCs)。此类细胞不表达任何谱系的标志,但是其具备类似T淋巴细胞的功能。根据与此类细胞相关的转录因子、表而表达的受体以及其活化后分泌的特征性细胞因子,可将ILCs分为三类,包括ILC1s、ILC2s和ILC3s。在健康个体体内,ILCs的量微乎其微,而在病理状态下可被活化而比例增加。支气管哮喘发作时,活化的ILC2 s分泌IL-13、IL-5等细胞因子,维持Th2极化状态,参与免疫损伤。在这些促炎细胞因子的作用下,使气道局部呈现气道高反应性,形成典型的哮喘症状。ROR α,是维甲酸相关的孤核受体家族的成员之一,其在各种生物体的多种组织均有表达。作为ILC2s的特征性转录因子,RORα在ILC2s分化增殖过程中以及功能的发挥方面发挥重要作用。目前的研究发现,当小鼠缺失ROR α时,其体内ILC2s的数量减少,并且对于寄生虫的免疫能力减弱。而缺乏ILC2s的小鼠经过继转移ILC2s,则小鼠的ILC2s功能可获重建。目的为了探讨ROR α于支气管哮喘发生发展中的作用,为临床哮喘病的治疗寻找新的途径。本研究在建立哮喘小鼠模型的基础上,分析ILC2s在模型鼠的分布,并经构建ROR α表达载体研究其对ILC2s的调控作用,探讨用于干预哮喘模型鼠的可能性。旨在为ROR α的应用研究奠定理论依据和试验基础。材料与方法(1)临床病例分析:流式细胞术检测支气管哮喘患者外周血单个核细胞中ILC2s的比例;RT-qPCR方法检测外周血单个核细胞中RORα、T-bet、GATA3、IL-33、IL-25、IL-13、IL-5、IL-4 的 mRNA 表达水平;ELISA 法测定血清中的IL-33、IL-13、IL-5的蛋白含量;直线回归法分析RORα的mRNA表达水平与ILC2s活性的相关性、RORα的mRNA表达水平与Th1、Th2细胞相关分子的相关性、ILC2s的特征性细胞因子mRNA表达水平与Th2细胞特征性细胞因子的相关性。(2)小鼠哮喘模型的诱导和ROR α表达载体的体内干预试验:1)构建RORα表达载体:无菌分离小鼠脾脏组织,运用RT-PCR从小鼠脾脏组织的mRNA中克隆出mROR α全长编码区的cDNA,并将该cDNA连接到PMD-18T载体,同时鉴定该载体序列。再定向克隆入腺病毒的穿梭载体pDC316-mCMV-EGFP,并与骨架载体PBHGloxdetalE1, 3Cre一同于HEK293内包装,并纯化腺病毒。2)小鼠哮喘模型的诱导及RORα表达载体的干预:建立OVA诱导的支气管哮喘模型。利用Ad-ROR α干预后。运用HE染色的方法观察肺组织病理学的变化;流式细胞仪分析模型鼠外周血ILC2s细胞比例;用细胞计数池计数BALF中炎症细胞的浸润情况;ELISA检测血清IL-33、IL-13、IL-5等细胞因子的含量;肺组织和支气管肺泡灌洗液中RORα、IL-33、IL-13、IL-5、IL-4和GATA3等mRNA表达水平用RT-qPCR法测定、肺组织中ROR α、ST2蛋白的表达以Western-blot 法检测。结果(1)支气管哮喘患者外周血中IILC2s的比例相对于正常对照者明显升高,且血清IL-33以及ILC2s的特征性细胞因子IL-13、IL-5的蛋白含量也随之升高;同时,外周血单个核细胞中 RORα、IL-33、IL-13、IL-5、IL-4、GATA3 的 mRNA表达水平也呈升高趋势,而Th1细胞的转录因子T-bet的mRNA则呈降低趋势。相关性分析结果显示,RORα与ILC2s呈正相关,IL-33亦与ILC2s的特征性细胞因子IL-13、IL-5、IL-4呈正相关。(2)成功克隆了小鼠RORα基因的全长序列,经测序无误。经定向克隆的方法,成功将RORα的全长基因克隆入pDC316-mCMV-EGFP,经过PCR、酶切分析和测序鉴定均无误。(3)腺病毒的包装:成功大量制备了携带小鼠ROR α的穿梭载体pDC316-RORα-mCMV-EGFP、骨架载体 PBHGloxdetalEl,3Cre,随后将穿梭载体 pDC316-RORα -mCMV-EGFP与骨架载体PBHGloxdetalE1, 3Cre 一同转染状态良好的HEK293细胞,经过纯化后,成功地包装出了高滴度的携带小鼠RORα的腺病毒。(4)小鼠哮喘模型的建立:用OVA成功诱导小鼠哮喘动物模型,经模型鼠肺组织HE染色镜检,可见大量炎症细胞浸润。与OVA组和OVA+Ad-EGFP组相比,Ad-RORα处理的模型鼠,其肺组织中的炎症细胞增多更为明显,且哮喘症状更为明显,出现强烈地抓耳挠腮、抬起前肢、大小便失禁、呼吸急促等哮喘表现。同时,处理组小鼠与未处理的OVA诱模组和Ad-EGFP处理组相比,其支气管肺泡灌洗液中IgE的水平明显增加。(5)哮喘模型中ILC2s相关的因子检测:Ad-RORα处理组循环ILC2s明显高于未处理的模型鼠和Ad-EGFP对照组,提示ILC2s参与的哮喘的发病过程,而转录因子RORα发挥了推波助澜的作用。此外,在Ad-RORα处理组,促进ILC2s活化的IL-33,ILC2s的特征性细胞因子IL-13、IL-5和转录因子GATA3,以及Th2细胞因子IL-4等均明显升高。同时,我们亦发现IL-33蛋白升高,升高的IL-33进一步促进ILC2s的增殖活化。结论哮喘患者机体ILC2s数量及其相关分子表达增加,与此同时,上调的RORα与ILC2s比例呈现明显的正相关关系。成功构建了 Ad-RORα病毒载体,成功建立了 OVA诱导的小鼠哮喘模型,并经体内试验表明了外源性ROR α对哮喘发生发展的免疫病理作用。
[Abstract]:Background bronchial asthma is a chronic inflammatory airway disease involving multiple immune cells, which can occur at any age. In recent years, more and more studies have found that there are a group of non T non B immune cells in the body of bronchial asthma, that is, innate lymphoid cells (ILCs). The mark of a line, but has a function similar to the T lymphocyte. According to the transcription factors associated with such cells, the expressed receptor and its activated secretory characteristic cytokine can be divided into three classes, including ILC1s, ILC2s and ILC3s. in healthy individuals, and the amount of ILCs is minimal and can be activated in a pathological state. When the asthma attacks, the activated ILC2 s secretes IL-13, IL-5 and other cytokines, which maintain Th2 polarization and participate in the immune injury. Under the action of these proinflammatory cytokines, airway hyperresponsiveness is localized in the airway, forming a typical asthma symptom.ROR alpha, which is one of the members of the retinoic acid related lone receptor family. It is expressed in various tissues of various organisms. As a characteristic transcription factor of ILC2s, ROR alpha plays an important role in the differentiation and proliferation of ILC2s and function. The present study found that when ROR alpha was missing in mice, the number of ILC2s in the body was reduced, and the immune ability of the parasites was weakened. But the lack of ILC2s was the lack of ILC2s. In order to explore the role of ROR alpha in the development of bronchial asthma in order to explore the role of ROR alpha in the development of bronchial asthma in order to find a new way for the treatment of asthma, the purpose of this study was to explore the distribution of ILC2s in the model mice and to construct the ROR alpha expression vector. Its role in regulating ILC2s and exploring the possibility of interfering with asthma model mice. The purpose is to lay a theoretical basis and experimental basis for the application of ROR alpha. Materials and methods (1) clinical case analysis: flow cytometry was used to detect the proportion of ILC2s in peripheral blood mononuclear cells of bronchial asthma patients; RT-qPCR method was used to detect peripheral blood mononuclear cells. The mRNA expression level of ROR alpha, T-bet, GATA3, IL-33, IL-25, IL-13, IL-5, IL-4; ELISA method for the determination of IL-33, IL-13, and protein content in serum; linear regression analysis of the correlation between the expression level and the activity, the correlation of the expression level with the cell related molecules and the characteristic cytokine. The correlation between the expression level of mRNA and the characteristic cytokines of Th2 cells. (2) the induction of mouse asthma model and the intervention test of ROR alpha expression vector in vivo: 1) construction of ROR alpha expression vector: aseptic isolation of mouse spleen tissue, and RT-PCR from the mRNA of spleen tissue of mice to clone cDNA of mROR alpha full length coding region, and connect the cDNA to PMD. -18T vector, simultaneously identified the vector sequence, then cloned into the shuttle vector pDC316-mCMV-EGFP of the adenovirus, and packed with the skeleton carrier PBHGloxdetalE1, 3Cre together in HEK293, and purified the adenovirus.2 mouse asthma model and the intervention of the ROR alpha expression vector: build a OVA induced bronchial asthma model. Use Ad-ROR alpha dry. Prognosis. HE staining was used to observe the changes in lung histopathology; flow cytometry was used to analyze the proportion of ILC2s cells in the peripheral blood of the rat model; the infiltration of inflammatory cells in BALF was counted with cell count pool; the content of IL-33, IL-13, IL-5 and other cytokines in serum were detected by ELISA; ROR a, IL-33, IL-13, IL-5 in lung tissue and bronchoalveolar lavage fluid The mRNA expression levels of IL-4 and GATA3 were measured by RT-qPCR, and the expression of ROR alpha and ST2 protein in lung tissue was detected by Western-blot method. Results (1) the proportion of IILC2s in peripheral blood of bronchial asthma patients was significantly higher than that of normal controls, and the serum IL-33 and ILC2s characteristic cytokines were IL-13, and the protein content of IL-5 was also increased. At the same time, the expression level of ROR alpha, IL-33, IL-13, IL-5, IL-4, GATA3 in peripheral blood mononuclear cells also increased, while the mRNA of Th1 cell transcriptional factor T-bet decreased. The correlation analysis showed that ROR alpha was positively related to ILC2s. (2) success (2) success The full-length sequence of the mouse ROR alpha gene was cloned and sequenced. The full length gene of ROR alpha was cloned into pDC316-mCMV-EGFP by directional cloning. Through PCR, the enzyme digestion analysis and sequencing were all correct. (3) the packaging of adenovirus: the successful preparation of a large number of shuttle carrier pDC316-ROR alpha -mCMV-EGFP carrying mouse ROR alpha, skeleton carrier PB HGloxdetalEl, 3Cre, then transfection of the shuttle carrier pDC316-ROR alpha -mCMV-EGFP and the skeleton carrier PBHGloxdetalE1, 3Cre into good HEK293 cells. After purification, the high titer carrying mouse ROR alpha adenovirus was successfully packaged. (4) the model of mice asthma was successfully induced by OVA, and the model was successfully induced by OVA. A large number of inflammatory cells were found in the HE staining of the lung tissue of the rat. Compared with the OVA and OVA+Ad-EGFP groups, the model rats treated with Ad-ROR alpha were more obvious in the lung tissue, and the symptoms of asthma were more obvious. The symptoms of strong scratches, forelimbs, incontinence, shortness of breath, and other asthma symptoms were raised. The level of IgE in the bronchoalveolar lavage fluid in the mice was significantly increased compared with the untreated OVA induced and Ad-EGFP treatment groups. (5) the ILC2s related factors in the asthma model were detected: the Ad-ROR alpha treatment group was significantly higher than the untreated model rat and the Ad-EGFP control group. The pathogenesis of ILC2s involved in asthma was presented, and the transcription factor RO was shown to be RO. R alpha played a contributing role. In addition, in the Ad-ROR alpha treatment group, IL-33 promoted ILC2s activation, ILC2s's characteristic cytokines IL-13, IL-5 and transcription factor GATA3, and Th2 cytokine IL-4 were all significantly increased. Meanwhile, we also found that IL-33 protein was elevated, and the increased IL-33 further promoted the proliferation and activation of the asthma. Conclusion asthma patients The number of ILC2s and its related molecules increased. At the same time, there was a positive correlation between the up regulation of ROR alpha and the proportion of ILC2s. The Ad-ROR alpha virus vector was successfully constructed and a OVA induced mouse model of asthma was successfully established, and the immunological pathological effect of exogenous ROR alpha on the development of asthma was demonstrated in vivo.
【学位授予单位】：江苏大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R562.25

【参考文献】

相关期刊论文 前3条

1 王琳;蔡婷;欧阳效晴;俎红丽;赵艳红;;褪黑激素核受体RORα的生理功能及其与绒毛生长关系的研究进展[J];中国畜牧杂志;2013年21期

2 李国豪;徐邦牢;雷秀霞;梁成结;王浩平;梁颜笑;;壁虎粉对哮喘豚鼠模型干预作用的实验研究[J];热带医学杂志;2007年02期

3 李志奎,王长征,钱桂生;地塞米松对哮喘豚鼠嗜酸细胞中白细胞介素3、5及粒-巨噬细胞集落刺激因子受体表达的影响[J];中华结核和呼吸杂志;2001年09期

，

本文编号：2058313