【作者】 黄江菊；

【导师】 朱道银；

【作者基本信息】 重庆医科大学 ， 内科学， 2004， 博士

【摘要】 目的 变应性鼻炎(allergic rhinitis，AR)和哮喘是严重影响人类健康的常见气道变态反应性疾病，近年来发病率呈上升趋势。目前缺乏有效的预防和根治措施。变应性鼻炎和哮喘是体外环境因素作用于机体导致异常免疫反应，造成Th1和Th2免疫反应失衡而引发的以鼻腔和支气管粘膜Th2免疫反应为主的变态反应性炎症。防治变应性鼻炎和哮喘的研究可着眼于调节机体Th1和Th2免疫平衡的免疫调节治疗策略。 IL-12是重要的免疫调节因子，能促进T细胞接触变应原时分化为Th1细胞，激发并维持Th1免疫反应，同时促进活化的Th1细胞和NK细胞产生的IFN-γ和IL-2，抑制Th2细胞因子的表达和IgE的合成，抑制Th2免疫反应。构建单链IL-12DNA质粒，是将小鼠的两个IL-12基因亚单位(p40和p35)经多肽链linker连接载入质粒，该IL-12融合基因质粒能在体内、外稳定表达有高水平生物活性的IL-12。单链IL-12DNA质粒直接在气道局部转染表达IL-12，可达到抑制呼吸道变态反应的目的。 <WP=9>早期感染对调节Th1和Th2免疫平衡具有重要意义。“卫生假说”认为儿童期感染性疾病过程的缺乏可使Th2型免疫机制增强，从而促发哮喘等变态反应性疾病。针对人体免疫系统发育中“免疫偏移”调节过程通常在5岁前完成，可推测在这一时期采取可控的微生物疫苗措施能有效地防治变应性疾病。BCG是牛型分枝杆菌减毒活疫苗，是Th1型免疫反应介导剂，能通过诱导Th1型免疫反应调节Th1/Th2平衡，抑制变态反应的发生。近年来在变态反应疾病的防治中备受关注。本研究应用重组IL-12真核表达质粒、非特异性免疫调节剂BCG干预防治变应性鼻炎和哮喘。观察IL-12真核表达质粒直接在气道局部转染对成年鼠气道变态反应炎症的干预，并观察IL-12真核表达质粒作为BCG基因佐剂对新生小鼠早期接种，抑制卵白蛋白致敏和激发的气道Th2免疫反应，抑制特异性IgE，从而抑制呼吸道变态反应炎症的发生。为变应性疾病疫苗的研究奠定基础，为临床防治变应性鼻炎、哮喘提供理论依据和实践方法。方法 1、将IL-12融合基因真核表达质粒pcDNA/IL-12(psIL-12)用PCR方法和DNA双向测序进行鉴定。将pcDNA3.1(+)、psIL-12转染COS-7细胞，通过RT-PCR、ELISA的方法以及斑点印迹法检测其在真核细胞中的表达。2、小鼠气道变态反应炎症模型制备：去鸡卵蛋白喂养的6周龄BALB/c小鼠，随机分为模型组和对照组。以OVA为变应原、以Al(OH)3<WP=10>为免疫佐剂，两次腹腔注射系统致敏、连续6次呼吸道雾化吸入OVA诱导气道变应性炎症，诱发变应性鼻炎、哮喘发生。取血清检测OVA特异性IgE抗体、行支气管肺泡灌洗(BAL)记录灌洗液(BALF)中嗜酸性粒细胞(EOS)百分比计数、留取鼻、肺组织标本观察变应性炎症病理改变。3、质粒制备：按照QIAGEN质粒大量抽提试剂盒说明书大量抽提pcDNA3.1质粒、psIL-12质粒。紫外分光光度计测定质粒纯度，核酸定量仪测定抽提质粒含量。溶于生理盐水调整浓度至1mg/ml。4、成年小鼠随机分组 ① psIL-12干预组；② pcDNA3.1(+)对照组； ③PBS对照组，每组10只。OVA复制气道变态反应炎症模型。第29天气道内应用单剂量的psIL-12、pcDNA3.1(+)、PBS/脂质体复合物。小鼠气管内注射质粒/脂质体复合物后24小时后取肺组织，RT-PCR检测肺单链IL-12的表达。激发后组织标本留取及形态学观察；BALF中及血中嗜酸性粒细胞(Eos)百分比计数；BALF上清中细胞因子IL-4、IFN-γ测定；血清中OVA特异性IgE抗体检测。5、清洁级BALB/c小鼠出生早期仔鼠随机分组 ① psIL-12接种组； ② BCG接种组；③ psIL-12+BCG接种组；④ 裸质粒对照组； ⑤ PBS对照组，每组8-12只。接种：出生48小时仔鼠分别皮内注射BCG单次，每次0.1ml(5×104CFU)，肌肉注射psIL-12及裸质粒每周一次，每次50μɡ，4次。小鼠体成熟(5周龄)后，OVA复制鼻变应性炎症模型。激发后组织标本留取及组织形态学观察；BALF中及血中嗜酸性粒细胞<WP=11>(Eos)百分比计数；BALF上清中细胞因子IL-4、IFN-γ测定；血清中OVA特异性IgE抗体检测。结果 1、鉴定IL-12融合基因真核表达质粒pcDNA/IL-12(psIL-12)构建成功；转染真核细胞并通过RT-PCR、ELISA以及斑点印迹法检测到细胞培养上清液中IL-12的表达。2、激发后模型组小鼠与对照组相比，肺部出现多种炎性细胞浸润；血清含高滴度IgE；BALF中EOS增加，BALF上清中白细胞介素-4(IL-4)升高、γ-干扰素(IFN-γ)降低。3、将PBS、pcDNA3.1(+)、psIL-12分别经气管内注射转染于气道变态反应炎症BALB/c小鼠，24小时后收集肺组织标本，通过 RT-PCR方法检测到psIL-12组小鼠肺组织内mRNA水平IL-12的有效表达；较之PBS对照组，实验组均能不同程度抑制气道变态反应炎性浸润，血清IgE下降，BALF中EOS减少，BALF上清中白细胞介素-4(IL-4)降低、γ-干扰素(IFN-γ)升高。以psIL-12组在肺内表达对气道变态反应的抑制最明显(P<0.05)。4、清洁级出生早期BALB/c仔鼠分别接种psIL-12、BCG、psIL-12+BCG、裸质粒和PBS对照；仔鼠体成熟后经OVA致敏和激发，psIL-12+BCG接种鼠BALF中IFN-γ含量显著高于BCG组；psIL-12、BCG、psIL-12+BCG组IL-4水平与PBS对照组比较有显著意义；组?更多还原

【Abstract】 OBJECTIVE: The prevalence of allergic rhinitis(AR) and asthma has increased steadily over the past 3 decades. They are two kinds of common allergic disease endanger the health of human beings and there is a lack of effective prevention and therapeutic measures. Abnormal immunological responses is induced by altered environmental conditions in cases of AR and asthma. The disequilibrium of Th1/Th2 touches off the allergic inflammation, especially Th2 immunologic response, in the nasal cavity and bronchial mucosa. Modulating the Th1/Th2 balance may represent a new strategy of preventing and curing AR and asthma. IL-12 is an important immunoloregulation factor,which strongly promotes the differentiation of Th1 cell, while the lymphocytes contacting <WP=14>with the allergen. Simultaneously, IL-12 maintains the Th1 allergic response and promotes the active Th1 cell and NK cell to excrete IFN-γ and IL-2, which suppress the expression of Th2 cytokines and the synthesis of IgE and reduce the Th2 allergic response.To gain the high and steady level of bioactivity of IL-12 both in vitro and vivo, we constructed a single-chain IL-12 DNA plasmid, in which the two IL-12 subunits(p35 and p40) were linked by a 54bp polypeptide linker and then transferred into the plasmid. The local transfer of the single-chain IL-12 DNA plasmid modifies the allergic inflammation in the airway.Early periodic infections play an important role in modulating the Th1/Th2 balance. Infection of tuberculosis or pertussis in childhood may improve the immune system,including total body and local pulmonary immune system, which can build up a Th1 immune response enviroment of Th1 profiles cytokines such as IFN-γ,IL-12 and TNF. Meanwhile, these Th1 cytokines suppress the function of Th2 cytokines and redress the disequilibrium of Th1/ Th2. BCG , an attenuated strain of living bovine tubercle bacillus and a inducer of strong Th1 immune response, could direct the response to Th1 and decrease the occurrence of allergic diseases.In this study, we investigated the intervention function of recombination IL-12 DNA and nonspecific immunoloregulation BCG in AR and asthma. we observed the intervention of recombination IL-12 in AR in mature <WP=15>mice and its association with BCG-inoculation at early stage to suppress the allergic inflammation in the OVA-induced allergic mouse models. This intervention could decrease the Th2 reaction and specific IgE and suppress the allergic inflammation in the airway. Our study may provide a new strategy in developing the vaccine of atopic diseases and the prevention of AR and asthma.METHODS:1.The expression of pcDNA/IL-12 in eukaryon cells is identified PCR and DNA sequencing. pcDNA3.1(+) and pcDNA/IL-12 were transfected into COS-7 cells and their expression in eukaryon cells were evaluated by PT-PCR,ELISA and Western Blot.2. 20 6-week-aged BALB/c mice were divided into cast and control groups by random. Mice were sensitized by intraperitoneal and subcutaneous injections of OVA and AL(OH)3 twice,then received 6 consecutive airway OVA by aerosolization to induce the airway inflammation, AR and asthma. The serum samples were obtained to determine the OVA-specific IgE antibody titers. The BALF were obtained through the BAL to determine the proportion of EOS. The pathological changes were observed in the pulmonary tissues and the expression of IL-4mRNA of lymphocytes in local airway were evaluated by immunohistochemistry. <WP=16>3. pcDNA3.1 and pcDNA/IL-12 plasmids were gained in great deal according to the QIAGEN introduction. The content of plasmids was evaluated by polynucleotide quantu machine and the purity by spectrophotometer. Plasmids were dissolved into NS to a concentration of 1mg/ml. 4.mature mice were divided into 3 groups by random: psIL-12, pcDNA3.1(+) control and PBS control. Each group contained 10 mice. All mice were sensitized and provoked with OVA to establish the animal models of airway inflammation. A single dose of psIL-12, pcDNA3.1(+), PBS-lipid complex was appli更多还原