本文選題：RORα + 支氣管哮喘�。� 參考：《江蘇大學(xué)》2017年博士論文

【摘要】：背景支氣管哮喘是一類由多種免疫細(xì)胞參與的慢性炎癥性氣道疾病,可發(fā)生于任何年齡。近年來,越來越多的研究發(fā)現(xiàn),支氣管哮喘患者體內(nèi)存在著一群非T非B的免疫細(xì)胞,即固有淋巴細(xì)胞(innate lymphoid cells, ILCs)。此類細(xì)胞不表達(dá)任何譜系的標(biāo)志,但是其具備類似T淋巴細(xì)胞的功能。根據(jù)與此類細(xì)胞相關(guān)的轉(zhuǎn)錄因子、表而表達(dá)的受體以及其活化后分泌的特征性細(xì)胞因子,可將ILCs分為三類,包括ILC1s、ILC2s和ILC3s。在健康個(gè)體體內(nèi),ILCs的量微乎其微,而在病理狀態(tài)下可被活化而比例增加。支氣管哮喘發(fā)作時(shí),活化的ILC2 s分泌IL-13、IL-5等細(xì)胞因子,維持Th2極化狀態(tài),參與免疫損傷。在這些促炎細(xì)胞因子的作用下,使氣道局部呈現(xiàn)氣道高反應(yīng)性,形成典型的哮喘癥狀。ROR α,是維甲酸相關(guān)的孤核受體家族的成員之一,其在各種生物體的多種組織均有表達(dá)。作為ILC2s的特征性轉(zhuǎn)錄因子,RORα在ILC2s分化增殖過程中以及功能的發(fā)揮方面發(fā)揮重要作用。目前的研究發(fā)現(xiàn),當(dāng)小鼠缺失ROR α?xí)r,其體內(nèi)ILC2s的數(shù)量減少,并且對(duì)于寄生蟲的免疫能力減弱。而缺乏ILC2s的小鼠經(jīng)過繼轉(zhuǎn)移ILC2s,則小鼠的ILC2s功能可獲重建。目的為了探討ROR α于支氣管哮喘發(fā)生發(fā)展中的作用,為臨床哮喘病的治療尋找新的途徑。本研究在建立哮喘小鼠模型的基礎(chǔ)上,分析ILC2s在模型鼠的分布,并經(jīng)構(gòu)建ROR α表達(dá)載體研究其對(duì)ILC2s的調(diào)控作用,探討用于干預(yù)哮喘模型鼠的可能性。旨在為ROR α的應(yīng)用研究奠定理論依據(jù)和試驗(yàn)基礎(chǔ)。材料與方法(1)臨床病例分析:流式細(xì)胞術(shù)檢測(cè)支氣管哮喘患者外周血單個(gè)核細(xì)胞中ILC2s的比例;RT-qPCR方法檢測(cè)外周血單個(gè)核細(xì)胞中RORα、T-bet、GATA3、IL-33、IL-25、IL-13、IL-5、IL-4 的 mRNA 表達(dá)水平;ELISA 法測(cè)定血清中的IL-33、IL-13、IL-5的蛋白含量;直線回歸法分析RORα的mRNA表達(dá)水平與ILC2s活性的相關(guān)性、RORα的mRNA表達(dá)水平與Th1、Th2細(xì)胞相關(guān)分子的相關(guān)性、ILC2s的特征性細(xì)胞因子mRNA表達(dá)水平與Th2細(xì)胞特征性細(xì)胞因子的相關(guān)性。(2)小鼠哮喘模型的誘導(dǎo)和ROR α表達(dá)載體的體內(nèi)干預(yù)試驗(yàn):1)構(gòu)建RORα表達(dá)載體:無菌分離小鼠脾臟組織,運(yùn)用RT-PCR從小鼠脾臟組織的mRNA中克隆出mROR α全長(zhǎng)編碼區(qū)的cDNA,并將該cDNA連接到PMD-18T載體,同時(shí)鑒定該載體序列。再定向克隆入腺病毒的穿梭載體pDC316-mCMV-EGFP,并與骨架載體PBHGloxdetalE1, 3Cre一同于HEK293內(nèi)包裝,并純化腺病毒。2)小鼠哮喘模型的誘導(dǎo)及RORα表達(dá)載體的干預(yù):建立OVA誘導(dǎo)的支氣管哮喘模型。利用Ad-ROR α干預(yù)后。運(yùn)用HE染色的方法觀察肺組織病理學(xué)的變化;流式細(xì)胞儀分析模型鼠外周血ILC2s細(xì)胞比例;用細(xì)胞計(jì)數(shù)池計(jì)數(shù)BALF中炎癥細(xì)胞的浸潤(rùn)情況;ELISA檢測(cè)血清IL-33、IL-13、IL-5等細(xì)胞因子的含量;肺組織和支氣管肺泡灌洗液中RORα、IL-33、IL-13、IL-5、IL-4和GATA3等mRNA表達(dá)水平用RT-qPCR法測(cè)定、肺組織中ROR α、ST2蛋白的表達(dá)以Western-blot 法檢測(cè)。結(jié)果(1)支氣管哮喘患者外周血中IILC2s的比例相對(duì)于正常對(duì)照者明顯升高,且血清IL-33以及ILC2s的特征性細(xì)胞因子IL-13、IL-5的蛋白含量也隨之升高;同時(shí),外周血單個(gè)核細(xì)胞中 RORα、IL-33、IL-13、IL-5、IL-4、GATA3 的 mRNA表達(dá)水平也呈升高趨勢(shì),而Th1細(xì)胞的轉(zhuǎn)錄因子T-bet的mRNA則呈降低趨勢(shì)。相關(guān)性分析結(jié)果顯示,RORα與ILC2s呈正相關(guān),IL-33亦與ILC2s的特征性細(xì)胞因子IL-13、IL-5、IL-4呈正相關(guān)。(2)成功克隆了小鼠RORα基因的全長(zhǎng)序列,經(jīng)測(cè)序無誤。經(jīng)定向克隆的方法,成功將RORα的全長(zhǎng)基因克隆入pDC316-mCMV-EGFP,經(jīng)過PCR、酶切分析和測(cè)序鑒定均無誤。(3)腺病毒的包裝:成功大量制備了攜帶小鼠ROR α的穿梭載體pDC316-RORα-mCMV-EGFP、骨架載體 PBHGloxdetalEl,3Cre,隨后將穿梭載體 pDC316-RORα -mCMV-EGFP與骨架載體PBHGloxdetalE1, 3Cre 一同轉(zhuǎn)染狀態(tài)良好的HEK293細(xì)胞,經(jīng)過純化后,成功地包裝出了高滴度的攜帶小鼠RORα的腺病毒。(4)小鼠哮喘模型的建立:用OVA成功誘導(dǎo)小鼠哮喘動(dòng)物模型,經(jīng)模型鼠肺組織HE染色鏡檢,可見大量炎癥細(xì)胞浸潤(rùn)。與OVA組和OVA+Ad-EGFP組相比,Ad-RORα處理的模型鼠,其肺組織中的炎癥細(xì)胞增多更為明顯,且哮喘癥狀更為明顯,出現(xiàn)強(qiáng)烈地抓耳撓腮、抬起前肢、大小便失禁、呼吸急促等哮喘表現(xiàn)。同時(shí),處理組小鼠與未處理的OVA誘模組和Ad-EGFP處理組相比,其支氣管肺泡灌洗液中IgE的水平明顯增加。(5)哮喘模型中ILC2s相關(guān)的因子檢測(cè):Ad-RORα處理組循環(huán)ILC2s明顯高于未處理的模型鼠和Ad-EGFP對(duì)照組,提示ILC2s參與的哮喘的發(fā)病過程,而轉(zhuǎn)錄因子RORα發(fā)揮了推波助瀾的作用。此外,在Ad-RORα處理組,促進(jìn)ILC2s活化的IL-33,ILC2s的特征性細(xì)胞因子IL-13、IL-5和轉(zhuǎn)錄因子GATA3,以及Th2細(xì)胞因子IL-4等均明顯升高。同時(shí),我們亦發(fā)現(xiàn)IL-33蛋白升高,升高的IL-33進(jìn)一步促進(jìn)ILC2s的增殖活化。結(jié)論哮喘患者機(jī)體ILC2s數(shù)量及其相關(guān)分子表達(dá)增加,與此同時(shí),上調(diào)的RORα與ILC2s比例呈現(xiàn)明顯的正相關(guān)關(guān)系。成功構(gòu)建了 Ad-RORα病毒載體,成功建立了 OVA誘導(dǎo)的小鼠哮喘模型,并經(jīng)體內(nèi)試驗(yàn)表明了外源性ROR α對(duì)哮喘發(fā)生發(fā)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.
【學(xué)位授予單位】：江蘇大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R562.25

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