【作者】 修晶辉；

【导师】 夏咸柱； 张连峰；

【作者基本信息】 北京协和医学院 ， 免疫学， 2014， 博士

【摘要】 背景及目的哮喘被定义为一种支气管气道慢性炎症,其特征为可逆性气道阻塞,粘液分泌增加和嗜酸性粒细胞、气道中性粒细胞、肥大细胞和T淋巴细胞浸润。在儿童和成人中哮喘是一种最常见的慢性肺部疾病,但是这种疾病是我们目前无法预防或治愈的。世界各地哮喘患病率流行病学调查结果发现儿童哮喘患病率3.3%-29%,成人哮喘患病率1.2%-25.5%,据估计日前全球约有3亿人罹患哮喘。全球哮喘病死率约1/10万,全世界约每年25万人死于哮喘,其中年轻人占很大比例。美国哮喘患者病死率从1979年到1998年20年间上升了2.5倍。我国儿童哮喘患病率调查发现儿童哮喘患病率由0.91%上升到].54%,较10年前上升了64.84%。我国现有的成人流行病学调查结果显示哮喘患病率存在地区性差异。据WHO估计每年由于哮喘而导致的调整伤残生命年(DALYs)达1500万,约占全球疾病负担的1%。在世界范围内计算,哮喘相关的经济花费比肺结核和艾滋病的总数还高。哮喘疾病带给各国政府、家庭及患者十分沉重的经济负担,同时哮喘疾病对患者的口常活动以及心理和社会功能都造成了很大的影响。白细胞介素35(IL-35)是山P35和Ebi3二个亚基构成的细胞因子,属于IL-12家族成员,由Treg细胞分泌的一种抗炎和免疫抑制的细胞因子,主要表现为促进抑制性T细胞的增生,促进抑制性细胞因子IL-10和TNF-β的分泌同时抑制促炎症细胞因子IL-17的表达。目前与IL-35相关的疾病研究主要集中在感染性疾病,变态反应性和自身免疫性疾病等几个方面。而小鼠哮喘模型中体外给予IL-35蛋白通过抑制Th17细胞从而抑制小鼠哮喘的发生,我们推测在IL-35转基因小鼠中过表达的IL-35有可能通过调节Treg细胞和Th17细胞等免疫细胞进而影响哮喘炎症的产生过程。研究IL-35可能为变态反应治疗提供重要线索,但是,目前尚无IL-35转基因小鼠体内变态反应调节作用的研究报道,本研究利用IL-35转基因小鼠、OVA刺激诱导的哮喘模型研究IL-35在变态反应中抑制炎症发生的作用及免疫调节机制。方法利用分子克隆技术及显微注射技术构建过表达IL-35转基因小鼠,采用western blotting技术检测IL-35转基因小鼠中IL-35细胞因子的两个亚基EBI3和p35、IL-12和IL-27的表达情况。利用reblot技术对比分析IL-35转基因小鼠和同窝阴性小鼠血液、脾脏和肌肉细胞中IL-35的两个亚基EBI3和p35是否在同一位置表达。利用流式细胞术对比分析IL-35转基因小鼠和同窝阴性小鼠血液和脾脏中Treg细胞和T17细胞所占的百分比。利用腹腔注射接种1%OVA和铝佐剂每周一次连续接种3周,并于第四周连续5%OVA雾化四次建立小鼠哮喘小鼠模型,通过肺功能检测小鼠气道高反应情况,对比分析IL-35转基因小鼠和同窝阴性小鼠中的哮喘症状。在OVA刺激诱导的小鼠模型中,通过肺部病理学观察IL-35转基因小鼠和同窝阴性小鼠支气管周围的炎性细胞侵润和粘液的渗出情况。利用流式细胞术对比分析IL-35转基因小鼠和同窝阴性小鼠肺盥洗液中Treg细胞和Th17细胞所占的百分比。结果利用上述方法在整体水平证实：(1)建立了IL-35过表达转基因小鼠,western blotting证明IL-35表达量高于IL-12和IL-27。IL-35过表达转基因小鼠外周血和脾脏中Treg细胞的百分比增高。(2)建立了OVA小鼠哮喘模型。利用这种模型证实,转基因过表达IL-35能够抑制小鼠气道高反应。减少了支气管周围炎症细胞的浸润和粘液的分泌,说明IL-35在体内能够抑制小鼠的哮喘症状。IL-35能够在体内外活化Treg细胞,抑制Th17细胞分泌IL-17。说明,IL-35抑制小鼠哮喘的重要通路是通过抑制促炎症IL-17细胞因子实现的。结论总结以上研究结果首次证实以下结论(1)IL-35具有明显的抑制哮喘的作用。这种作用是通过促进Treg细胞和抑制Th17细胞的增殖和活化实现的。(2)IL-35通过促进Treg细胞增殖抑制细胞因子IL-17分泌的途径抑制哮喘炎症的发生和粘液的分泌。综上,过表达IL-35明显改善了小鼠哮喘的症状,主要是通过抑制促炎症细胞因子的产生而发挥作用的。人肠道病毒71感染小鼠模型时坏死性肌炎导致的限制性通气不足背景在小孩人肠道病毒(EV71)感染与手足口病(Hand, foot and mouth disease HFMD)的高度流行密切相关,并且偶尔能引起致死性并发症。但是大多数感染是自限的,引起的并发症包括：无菌性脑膜炎,乙型脑炎,脊髓灰质炎样急性弛缓性麻痹和神经起源的肺水肿或出血,上述是引起致死性症状的主要原因,然而它们的致病机理尚不清楚。因此推测坏死性肌炎引起的肺换气不足是否是引起小鼠死亡的原因之一。方法利用腹腔注射EV71病毒接种两周龄的ICR小鼠,观察小鼠大体的情况,体重称重和生存期观察,荧光定量PCR(qRT-PCR)检测被感染小鼠组织器官的病毒载量,病理学观察和免疫组化分析骨骼肌中炎症侵润和病毒载量情况,利用肺功能检测仪分析病毒感染组和对照组的吸气时间,呼吸频率,总的潮气量和呼吸容积的情况。结果本研究对两周龄的ICR小鼠感染EV71的小鼠适应株后,感染的小鼠表现为进行性麻痹并与感染后12天内死亡。由于EV71主要在小鼠的骨骼肌复制并引起严重的坏死性肌炎,然而在脑和其他组织中尚未发现损伤。结论呼吸相关的肌肉产生的坏死性肌炎引起严重的限制性肺通气不足和缺氧被认为是EV71感染小鼠模型后致死的主要原因,暗示在EV71感染的小鼠中坏死性肌炎可能是麻痹和致死的一个因素而不是中枢神经系统损伤引起的。人SCARB2蛋白提高人肠道病毒71型对转基因小鼠的感染力背景目地人肠道病毒71型(EV71)是微核糖核酸病毒科肠病毒属肠病毒种A型RNA病毒,能引起6岁以下儿童的手足口病。但是许多EV71感染会自发的恢复,然而EV71病毒感染偶尔伴随严重的并发症,包括脑干脑炎、无菌性脑膜炎、肺水肿或肺出血、急性弛缓性麻痹和心力衰竭等甚至死亡。病毒受体在病毒感染的早期起着决定性作用,主要有宿主范围和组织嗜性决定,细胞实验证实人清道夫受体B2(hSCARB2)是人类EV71的受体之一,本研究拟通过建立人SCARB2转基因小鼠,建立感染能力更高的小鼠模型。方法构建CMV启动子的SCARB2转基因表达载体,通过显微注射法建立C57BL/6J背景的人SCARB2转基因小鼠,PCR筛选阳性首建鼠。通过Western Blot和免疫组化检测目的蛋白在各组织中的表达。EV71感染转基因小鼠后,通过实时荧光定量PCR和免疫组化检测目的蛋白表达对病毒感染的促进效果。结果人SCARB2蛋白主要在转基因小鼠的骨骼肌和脑组织中表达,与野生型小鼠相比,转基因小鼠组织中的病毒载量显著提高4到5倍。结论人SCARB2的体内表达可促进EV71对转基因小鼠的感染,该蛋白在体内具有EV71受体功能。更多还原

【Abstract】 Backgroud and ObjectiveAsthma is defined as a chronic inflammation of the bronchial airways and characterized by reversible airway obstruction, which increased mucus production and infiltration of the airway with eosinophils, neutrophils, mast cells and T-lymphocytes. Asthma is one of the most common chronic lung diseases in children and adults, but it is a disease that we are currently unable to prevent or cure. Worldwide epidemiological survey found that prevalence rate of children asthma is3.3%～29%and prevalence rate of adult asthma is1.2%-25.5%, it was about3hundred million people suffer from asthma in the worldwide. Global asthma death rate was about1/100000, there were about250000people die of asthma each year worldwide, which is a large proportion of young people. America asthma mortality rate increased by2.5times from1979to1998years. Our children asthma prevalence survey found that children asthma prevalence rate increased from0.91%to1.54%, up64.84%between ten years. Adult epidemiological survey showed that asthma prevalence have regional difference. According to WHO estimates that asthma disability adjusted life years (DALYs) reached15000000each year, about1%in the global burden of disease. In the world, the economy of asthma related cost far more than the total number of pulmonary tuberculosis and AIDS, it is very heavy economic burden for governments, families and patients, at the same time, it is a great impact for the patient’s daily activities and patient’s the psychological and social function.Interleukins35(IL-35) is composed of P35subunit and Ebi3subunit, it is a kind of anti-inflammatory and immune suppression of cytokine belong to the IL-12famimlies and secreted by Treg cells.Its main performance for promoting Inhibitory T cell proliferation, promoting inhibitory cytokine TNF and IL-10-β secretion and inhibiting the expression of proinflammatory cytokines IL-17. Currently, IL-35related diseases research mainly focused on infectious diseases, allergic and autoimmune diseases. Nevertheless, IL-35cytokine can promote syndrome of asthma model in mice by inhibiting numbers of Th17cells in vitro, we hypothesized that overexpression of IL-35may be adjusting the immune cells numbers of Treg cells and Th17cells in IL-35transgenic mice, which affect the inflammation process of asthma. It is likely to provide important clues for allergy treatment by studying the regulation effect of IL-35in allergic reaction.However, there is no research reports about of allergic regulating effect in IL-35transgenic mice, In this study,we used IL-35transgenic mice and asthma model challenged by OVA to study the inflammation role of IL-35in the suppression of allergic respect and regulating mechanism on the immunization.MethodsUsing molecular cloning techniques and microinjection technology to build overexpression IL-35transgenic mice, using western blotting technique to detect the expression of EBI3subunit and p35subunit of IL-35, the expression of IL-12and IL-27in IL-35transgenic mice and negative littermates (NTG) mice respectively. Analysis the distribution of EBI3subunit and p35subunit of IL-35in blood, spleen and muscle cells of IL-35transgenic mice and negative littermates mice by using western reblot. Quantitatively analyzed the percentage of Treg cells and Thl7cells blood, spleen and pulmonary washing lotion in IL-35transgenic mice and negative littermates mice by flow cytometry. In order to establish mouse model of asthma in IL-35transgenic mice and negative littermates mice, we continuously intraperitoneally inoculated with1%OVA and aluminium adjuvant once a week for3weeks, and the fourth week in a row with four days challeng by5%OVA atomization. Detected airway hyperresponsiveness of mouse model of asthma by the pulmonary function testing, the comparativly analysize asthma syndrome of IL-35transgenic mice and nontransgenic mice. Pathology observed the inflammatory cell exudation and bronchial mucus infiltration of lungs in OVA-challenged IL-35transgenic mice and OVA-challenged negative littermates mice. Comparativly analysize the percentage of Treg cells and Thl7cells of pulmonary washing lotion in mouse model of IL-35transgenic mice and negative littermates mice by flow cytometry.ResultsThe results of the experiments in vivo supported that:(1) Established transgenic over-expression of IL-35, IL-35expression quantity higher than that of IL-12and IL-27was proved by western blotting, the percentage of Treg cells increased in peripheral blood and spleen in overexpression IL-35transgenic mice.(2) Established mouse model of asthma. It confirmed that transgenic overexpression of IL-35can inhibit airway hyperresponsiveness in mice by using this model. Reduced the inflammatory cells infiltration and mucus secretion in the surrounding of bronchus, this explain that IL-35can inhibit mouse asthma symptoms in vivo. IL-35can activate the Treg cells in vivo, inhibit the secrete of IL-17by inhibiting the numbers of Th17cells, the signaling pathway IL-35inhibit mouse asthma is one of the important pathways by inhibiting proinflammatory IL-17cytokine.ConclusionsTaken togerther, our results indicated that:(1) IL-35has obvious inhibitory effect in mouse asthma.It affect by promoting Treg cells and inhibiting proliferation and activation of Th17cells.(2) IL-35could inhibit inflammation and mucus secretion in mouse astnma by promoting Treg cell proliferation and inhibiting the secretion of IL17cytokine. In conclusion, overexpression of IL-35can significantly improve the mouse asthma symptoms, it play the role in mainly inhibiting production of proinflammatory cytokine. Necrotizing myositis caused restrictive hypoventilation in human enterovirus71infected-mouse modelBackgroud and ObjectiveEV71infection is associated with a high prevalence of Hand, foot and mouth disease (HFMD) in children and occasionally causes lethal complications. Most of the infections were self-limited, while the complications including aseptic meningitis, encephalitis or poliomyelitis-like acute flaccid paralysis, and neurological originated pulmonary edema or hemorrhage were the main causes of lethal symptoms, the pathogenesis of which were remained to be clarified. Hence we presumed that caused hypoventilation caused by Necrotizing inflammation is one of the causes of death in mice.MethodsEV71virus inoculated two weeks old ICR mice by intraperitoneal (i.p.) injection, then we observe situation of the mice body generally, the weight of the mice bady and survival situation of mice, detecting infected viral load of tissues and organs by fluorescence quantitative PCR (qRT-PCR), pathological and immunohistochemical analysis inflammation and viral load of skeletal muscle in infected mice, we can analyze inspiratory time, respiratory rate, tidal volume and respiratory total volume of virus infection group of mice and control group of mice by pulmonary function detector.Results In present study, two-week-old ICR mouse were infected with a mouse-adapted EV71strain, and then the infected-mice demonstrated progressive paralysis and died within12days post infection. EV71mainly replicates in skeletal muscle tissues and caused severe necrotizing myositis, whereas the lesion in CNS and other tissues was not observed.ConclusionsThe necrotizing myositis of respiratory-related muscles caused severe restrictive hypoventilation and subsequently hypoxic, which was supposed as one reason of death for the EV71-infected mouse, suggesting that necrotic myositis caused may be a reason of paralysis and death beside of CNS injury in EV71-infected mice. Human SCARB2enhanced the infection of human enterovirus71on transgenic miceBackgroud and ObjectiveHuman enterovirus71(EV71) is a RNA virus of the micro RNA virus enterovirus, it can cause hand, foot and mouth disease in children under6years old.But some of EV71infections will spontaneous recovery, and EV71infection accompany serious complications occasionally, including the brain stem, aseptic meningitis, encephalitis, pulmonary edema and pulmonary hemorrhage, heart failure and acute flaccid paralysis and even death.the virus receptors play a pole role in the early days ofthe virus infection, mainly include host range and tropism of tissue.Human SCARB2was identified as one of the functional receptors of EV71in vitro, so establish more higher infection ability transgenic mice by establishment of hunman SCARB2transgenic mice in present study.MethodsThe transgenic vector was constructed by inserting the human SCARB2gene under the CMV promoter and then were subjected to establish transgenic mice by microinjection. the genotype of transgenic line was identified by PCR and the expression level of target protein was detected by Western blot. Viral load in the tissues of transgenic mice was detected by immunohistochemical staining and quantitative real-time PCR.Results One line of transgenic mice in C57BL/6J background with high levels of SCARB2expression in skeletal muscle and brain was identified. Upon infection with EV71, the virus burden of4to5times in muscle and brain of transgenic mice were significantly higher than that of wild type mice.ConclusionhSCARB2is a functional receptor of EV71in vivo, as expression of it could promote the infection of EV71on transgenic mice.更多还原