本文選題：變應性鼻炎 + siRNA�。� 參考：《重慶醫(yī)科大學》2015年博士論文

【摘要】：目的：變應性鼻炎(allergic rhinitis, AR)是發(fā)生在鼻粘膜的變態(tài)反應性疾病,其發(fā)病以兒童和青壯年居多。據(jù)WHO近年公布的數(shù)據(jù)表明全世界目前超過5億人罹患此病。流行病學資料也顯示,在2008年美國變應性鼻炎的發(fā)病率高達16.7%,在慢性病中居第5位,而AR也是兒童最常見的慢性疾病,每年用于診治變應性鼻炎的費用高達數(shù)十億美元。而在我國,2007年公布的國內(nèi)11個中心城市的流行病學資料,成人自報患病率介于9-24.6%,兒童則介于1.8-13.67%。變應性鼻炎雖不會引起嚴重后果,但能很大程度影響患者日常生活質(zhì)量。目前,對變應性鼻炎最常用的治療方案是鼻噴糖皮質(zhì)激素和口服抗組胺藥,該方法雖然能快速控制鼻過敏性炎癥,有效緩解臨床癥狀,卻不能阻止變應性鼻炎反復發(fā)作,距治愈的目標尚遠。研究表明,僅有約60%的患者對變應性鼻炎的治療總體上感到非常滿意,且隨著時間的推移,藥物治療的療效會逐漸減弱,這些治療上的困難究其原因在于變應性鼻炎的發(fā)病機制尚未完全明了,難以探索出更為有效的治療手段。因此,加大對變應性鼻炎發(fā)病機制的研究和防治措施的探討是目前耳鼻咽喉科學領域高度重視和急待解決的重點之一。變應性鼻炎的病因尚不明確,一般認為與特應性個體、遺傳學背景及環(huán)境因素有關。大量的實驗研究證實,鼻變態(tài)反應的發(fā)病機制是以Th2反應為主的變態(tài)反應性疾病,即’Th1/Th2失衡引發(fā)的下游生物效應導致變應性鼻炎的發(fā)生,但最近隨著研究的進一步深入,有些學者提出新識別的T細胞亞群可能對Th2細胞的分化有調(diào)節(jié)作用,如CD4+CD25+調(diào)節(jié)性T細胞對Th2細胞所介導的呼吸道變應性炎癥發(fā)揮強大的抑制作用。變應性個體可能存在Treg/Th2失衡,但其復雜的信號傳導途徑和機制仍在研究和探索中,這必將為變應性鼻炎提供新的治療靶點。DC是體內(nèi)功能最強大的專職抗原提呈細胞,也是唯一能使初始T淋巴細胞活化的APC,在免疫系統(tǒng)中處于啟動、調(diào)控并維持免疫應答的中心環(huán)節(jié)。有研究證實變應性鼻炎體內(nèi)Th2的極化狀態(tài)與DC細胞的功能異常有關,DC成為研究影響T細胞活化與增殖的重要靶細胞。未成熟DC由于其表面缺乏共刺激因子,在體內(nèi)或體外均顯示出誘導Treg細胞的特性,因此采用基因修飾的方法對DC加以修飾,使其保持穩(wěn)定的未成熟DC的特性,從而達到抑制效應T細胞的活化而增強Treg細胞活化的目的。本課題擬以AR為研究對象,闡明AR中是否存在Treg/Th2失衡并以DC-Th細胞間相互作用為主線,以慢病毒載體CD86-siRNA對DC的調(diào)控為切入點,闡述AR中基因修飾DC對Treg/Th2失衡的調(diào)控,說明Th失衡的上游事件及其免疫調(diào)控因素,為AR發(fā)病機制的研究提供新的思路。方法：1、納入排除標準：本研究嚴格按照中華醫(yī)學會變應性鼻炎診斷和治療指南(2009,武夷山),所有入組患者均來自重慶醫(yī)科大學附屬兒童醫(yī)院耳鼻咽喉科門診,均為首次診斷,未接受變應性鼻炎相關治療,對照組排除有感染性的、占位性的、藥物誘導性鼻炎和有任何并發(fā)癥的患者,在獲得其家長知情同意后,將其納入研究對象。2、探討AR患者與正常對照外周血中Treg/Th2細胞亞群分布及與臨床癥狀的關系、Treg/Th2相關細胞因子及轉(zhuǎn)錄因子的表達差異。(1)按照ELISA試劑盒操作步驟檢測AR患者與正常對照外周血中總IgE、IL-4、IL-5、TGF-β1的含量。(2) RT-PCR擴增檢測外周血轉(zhuǎn)錄因子FOXP、GATA-3mRNA表達情況。(3)采用Western Blot方法檢測AR患者及正常對照組外周血PBMC中FOXP3、GATA-3蛋白水平。3、慢病毒載體siRNA序列轉(zhuǎn)染人樹突狀細胞沉默CD86基因?qū)reg/Th2細胞分化的影響(1)慢病毒載體siRNA的構(gòu)建(2) MACS分選人外周血CD14+單核細胞并誘導成熟DC(3) MACS分選人外周血CD4十T細胞(4)攜帶特異性siRNA的慢病毒轉(zhuǎn)染DC(5)AR組未轉(zhuǎn)染DC、轉(zhuǎn)染后DC、正常對照組DC與外周血CD4+T細胞共培養(yǎng)將未轉(zhuǎn)染DC、轉(zhuǎn)染后DC與外周血CD4+T細胞共培養(yǎng),按照ELISA試劑盒操作步驟檢測共培養(yǎng)上清液中IL-4、IL-5、TGF-β1的含量,RT-PCR法檢測FOXP3、GATA-3mRNA水平,Western Blotting方法檢測FOXP3、GATA-3蛋白水平,以評價CD86基因修飾后DC對Th細胞分化的影響。結(jié)果：(1)AR組與對照組相比,血清總IgE水平、嗜酸性粒細胞比例、T5SS均明顯升高,且血清總IgE水平、嗜酸性粒細胞比例與T5SS呈正相關,而TGF-β1/IL-4、TGF-β1/IL-5、FOXP3 mRNA/GATA3 mRNA、FOXP3 protein/GATA3 protein比值與T5SS呈負相關。(2) ELISA檢測AR血清中IL-4, IL-5, TGF-β1的表達,AR組與對照組相比,IL-4,IL-5明顯增高,而TGF-β1的水平AR組明顯低于對照組。RT-PCR檢測Treg轉(zhuǎn)錄因子FOXP3 mRNA的表達,AR組明顯低于對照組,而AR組Th2轉(zhuǎn)錄因子GATA3 mRNA的表達明顯高于對照組。Western blotting檢測FOXP3與GATA3蛋白表達水平,結(jié)果GATA3, FOXP3蛋白表達趨勢與二者mRNA的表達趨勢一致。(3)應用MACS法從血液中成功提取CD14+單核細胞并刺激轉(zhuǎn)化成熟DC,成功提取CD4+T淋巴細胞,建立共培養(yǎng)體系(DC/CD4+T1:4);應用CD86-siRNA慢病毒成功轉(zhuǎn)染DC,MOI值20,轉(zhuǎn)染率約為60.2%；轉(zhuǎn)染后DC表面分子標志CD86的表達明顯下降；AR組未轉(zhuǎn)染,轉(zhuǎn)染后DC及正常對照組DC分別與CD4+T共培養(yǎng),上清液中Th2型細胞因子IL-4、IL-5在siRNA轉(zhuǎn)染后其表達水平明顯低于未轉(zhuǎn)染組,但仍明顯高于正常對照組,而Treg型細胞因子TGF-β1在AR未轉(zhuǎn)染組的表達低于正常對照,siRNA轉(zhuǎn)染后其表達明顯高于未轉(zhuǎn)染組,但仍低于正常對照組；Th2型轉(zhuǎn)錄因子GATA3 mRNA水平及蛋白表達水平siRNA轉(zhuǎn)染后明顯低于未轉(zhuǎn)染組,但高于正常對照組,而Treg型轉(zhuǎn)錄因子FOXP3 mRNA水平及蛋白表達水平,AR組轉(zhuǎn)染后表達高于未轉(zhuǎn)染組,二組均低于正常對照組。結(jié)論：(1)AR患者體內(nèi)FOXP3 mRNA和蛋白表達降低,而GATA3 mRNA和蛋白表達升高,FOXP3 mRNA/GATA3 mRNA、FOXP3蛋白/GATA3蛋白與AR的疾病嚴重程度(T5SS)呈負相關,提示AR患者體內(nèi)可能存在另外一種免疫失衡即Treg/Th2失衡。(2)通過體外細胞共培養(yǎng)實驗,應用攜帶特異性CD86-siRNA序列慢病毒載體轉(zhuǎn)染人樹突狀細胞沉默CD86基因,結(jié)果顯示樹突狀細胞表面分子CD86的表達明顯減少,這種穩(wěn)定的、未成熟DC影響了T細胞的分化,表現(xiàn)為Treg表達升高而Th2細胞表達降低,提示AR患者可以通過基因修飾DC來調(diào)節(jié)T細胞的活化,即改變Treg/Th2平衡,從而說明DC-Th軸在AR中的作用,為治療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.
【學位授予單位】：重慶醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R765.21

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3 陳小飛;變應性鼻炎如何防治？[N];健康時報;2008年

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5 湖北 副主任醫(yī)師 曾文;何為變應性鼻炎[N];家庭醫(yī)生報;2005年

6 蘇楠 林江濤;支氣管哮喘合并變應性鼻炎應同防同治[N];中國中醫(yī)藥報;2004年

7 于峰;變應性鼻炎怎么治？[N];大眾衛(wèi)生報;2007年

8 ;益肺通竅法治療變應性鼻炎[N];中國中醫(yī)藥報;2003年

9 副主任藥師 趙民生　(曹秀虹);變應性鼻炎的藥物治療[N];醫(yī)藥經(jīng)濟報;2009年

10 記者 李衛(wèi)中　通訊員 任曉輝;我國對變應性鼻炎規(guī)范化診斷和防治體系的研究正式啟動[N];山西經(jīng)濟日報;2008年

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5 于洋;鹿鵝鼻炎方對變應性鼻炎模型大鼠Th1/Th2細胞及相關細胞因子影響研究[D];北京中醫(yī)藥大學;2016年

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7 孫榮;從DC-Th軸探討CD86-siRNA基因修飾樹突狀細胞在變應性鼻炎中的作用研究[D];重慶醫(yī)科大學;2015年

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2 付敬敏;西寧地區(qū)變應性鼻炎患病情況及其相關因素分析[D];青海大學;2012年

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