胸腺基质淋巴细胞生成素、OX40和OX40受体在过敏性腹泻小鼠大肠黏膜中表达的关系

目的 建立小鼠过敏性腹泻模型,检测其大肠黏膜中胸腺基质淋巴细胞生成素(TSLP)、OX40、OX40受体(OX40L)的表达和肠系膜淋巴结细胞培养上清中IL-4、IFN-γ的水平,分析其之间的关系,探讨TSLP、OX40、OX40L在过敏性腹泻中的作用.方法 雌性BALB/c小鼠20只随机分成两组即对照组和实验组.采用酶联免疫吸附法(ELISA)检测肠系膜淋巴结细胞培养上清中IL-4和IFN-γ的水平,取大肠组织HE染色观察大肠组织病理改变,SP免疫组织化学技术检测TSLP、OX40、OX40L的表达.结果 ①HE染色结果显示:实验组比对照组小鼠结肠黏膜组织非特异性炎症反应显著,上皮排列不规则,有大量炎性细胞浸润,固有层可见大量嗜酸性粒细胞浸润;②TSLP、OX40、OX40L在过敏性腹泻小鼠大肠黏膜中的表达水平高于对照组的表达水平(t=7.07,t =7.81,t =7.79,P均＜0.01);③Spearman等级相关分析发现TSLP、OX40、0X40L三者间表达强度存在正相关(r=0.889,r=0.932,r=0.943,P均＜0.01),同时三者与肠系膜淋巴结细胞培养上清中IL-4水平呈正相关(r=0.891,r =0.936,r=0.886,P均＜0.05),而与IFN-γ水平呈负相关(r=-0.829,r=-0.881,r=-0.937,P均＜0.05).结论 TSLP、OX40、OX40L三者间表达存在正相关,并诱导了Th1/Th2轴向Th2轴漂移,促进了过敏性腹泻的发生发展.     

白三烯受体拮抗剂对呼吸道合胞病毒肺炎血清Th1/Th2细胞因子水平影响的研究

目的探讨白三烯受体拮抗剂对呼吸道合胞病毒肺炎患儿血清中Th1／Th2细胞因子、半胱氨酰白三烯水平的影响，研究降低呼吸道合胞病毒肺炎发展为哮喘的有效干预途径。方法将37例呼吸道合胞病毒肺炎患儿分为白三烯受体拮抗剂干预组：顺尔宁，4mg qd，疗程12周，糖皮质激素吸入干预组：出院后给予糖皮质激素吸入，200μg qd／bid，疗程12周。两组患儿入院后24h内及疗程结束用酶联免疫法检测CysLTs、IFN-γ、IL-4。另选10例健康同龄儿童血清标本作对照。结果呼吸道合胞病毒肺炎患儿血清中Th1／Th2细胞因子表达存在失衡，血清CysLTs水平明显高于健康对照组（t=7．85，P〈0．05）；顺尔宁干预组和糖皮质激素吸入干预组均能纠正Th1／Th2细胞因子失衡，差异无统计学意义（P〉0．05）。糖皮质激素吸入干预组血清CysLTs水平治疗前后无明显变化，差异无统计学意义（P〉0．05），顺尔宁干预组血清Cys仉水平治疗后明显下降，差异有统计学意义（P〈0．05）。结论呼吸道合胞病毒肺炎患儿存在Th1／Th2细胞因子失衡、血清CysLTs水平较健康同龄儿童明显升高，与哮喘有相似之处。白三烯受体拮抗剂早期干预能纠正Th1／Th2细胞因子失衡，降低血清CysLTs水平。     

Apyrase减轻过敏性哮喘小鼠气道炎症及气道重塑

目的探讨apyrase(三磷酸腺苷二磷酸水解酶)对过敏性哮喘小鼠气道炎症及气道重塑的作用。方法采用C57BL/6雌性小鼠建立鸡卵清蛋白(OVA)致敏和激发的过敏性哮喘模型,并给予apyrase处理;以非致敏的同背景雌性小鼠作为对照。于最后1次激发24~48 h,完成小鼠气道高反应性测定、支气管肺泡灌洗及取肺组织。病理学方法评价肺组织气道炎症、杯状细胞及气道重塑,瑞氏吉姆莎染色并计数灌洗液的分类细胞,酶联免疫吸附法测定灌洗液细胞因子,实时定量PCR法测定肺组织转录因子(GATA3)水平。结果 Apyrase减轻哮喘小鼠肺组织嗜酸性粒细胞炎症,降低气道炎性细胞、Th2相关细胞因子及肺组织GATA3核酸水平;减轻气道上皮杯状细胞增生及胶原沉积。结论 Apyrase可减轻小鼠过敏性哮喘的气道炎症及气道重塑。     

过敏性哮喘患者调节性T细胞对Th17细胞和Th9细胞的影响

目的:深入研究过敏性哮喘病人(轻到中度)急性发作期的调节性T细胞对Th17和Th9细胞功能影响的异常。方法:招募30例过敏性哮喘病人(轻到中度)急性发作期和30例健康者,采集外周静脉血30 ml,分离PBMC,然后应用磁珠法体外分离CD4+CD25+CD127-/low调节性T细胞(Tregs)和CD4+CD25-效应T细胞(选取分离纯度90%以上的标本继续下一步试验)。在PHA刺激下体外培养,检测效应T细胞增殖情况和Th17细胞、Th9细胞特异性基因(RORC和PU.1)表达及特异性细胞因子(IL-17和IL-9)分泌情况,检测哮喘病人Th17细胞和Th9细胞的异常情况;并给予Tregs干预,检测Tregs对Th17细胞和Th9细胞分化的影响。结果:哮喘组和健康对照组的Tregs均可以抑制CD4+CD25-效应细胞增殖反应,但哮喘组Tregs的抑制功能较健康对照组明显下降(P=0.03)。哮喘病人RORC表达和IL-17分泌均高于对照组(P0.05),Tregs对RORC表达的抑制能力在哮喘组有所下降(P0.05),但对IL-17分泌的抑制能力在哮喘组和对照组没有显著差别(P0.05);哮喘病人IL-9分泌无论是否加入Tregs干预均高于对照组(P0.05),但PU.1表达只有在加入Tregs干预后才高于对照组(P=0.04);Tregs对PU.1表达和IL-9分泌的抑制能力在哮喘组和对照组没有显著差别(P0.05)。结论:过敏性哮喘病人(轻到中度)急性发作期Tregs数量和功能下降,而Th17细胞和Th9细胞的特异性基因表达和特异性细胞因子生成增加,哮喘病人的Tregs在体外对Th17细胞的抑制功能有所下降,但尚未发现Tregs对Th9细胞的抑制功能明显降低。     

白三烯受体拮抗剂治疗反复发作的儿童过敏性紫癜的临床探讨

目的观察白三烯受体拮抗剂孟鲁司特治疗反复发作的儿童过敏性紫癜（HSP）的临床疗效。方法选择2008年2月-2011年2月我院收治的48例反复发作的儿童过敏性紫癜（1ISP）患儿随机分为两组,对照组（23例）采用常规治疗,治疗组（25例）在常规治疗基础上加服白三烯受体拮抗剂孟鲁司特（2—6岁为4mg／d、6—13岁为5mg／d,睡前服,共1个月）。结果治疗组总有效率（92％）明显高于对照组（60．8％）,两组相比,P〈0．05。差异有统计学意义。结论白三烯受体拮抗剂盂鲁司特治疗反复发作的儿童过敏性紫癜（HSP）疗效较好。     

哮喘小鼠模型肺组织nuocytes和Th9相关分子受体表达水平的变化及意义

目的分析nuocytes转录因子RORα及其分泌的细胞因子受体特异性结合链和Th9特征性细胞因子在哮喘小鼠肺组织表达的变化,探讨nuocytes对Th9细胞极化的影响,了解哮喘时肺组织对nuocytes和Th9细胞的应答状态。方法采用qRTPCR法检测RORα、IL-4Rα、IL-5Rα、IL-13Rα1、IL-13Rα2、IL-9和IL-9R等m RNA表达水平;对2类细胞特征性细胞因子受体表达水平的相关性及其与Ig E的表达水平的关系进行统计学分析。结果在哮喘小鼠模型中,RORα、IL-5Rα、IL-13Rα2和IL-9、IL-9R的m RNA表达水平均明显升高;相关性分析发现,IL-9及其受体的m RNA水平与RORα、IL-5Rα和IL-13Rα2 m RNA水平呈明显正相关,肺组织总Ig E的水平也随之增加。结论在哮喘小鼠肺组织中nuocytes和Th9相关分子及其受体优势表达,反映肺组织对2类细胞的积极应答状态,其在哮喘炎症中的作用由此可见一斑。     

组胺受体拮抗剂可防治哮喘豚鼠气道重塑和酸碱平衡紊乱

目的:探讨组胺受体拮抗剂对哮喘豚鼠气道重塑和酸碱平衡紊乱的防治作用。方法:将豚鼠分为正常对照组、哮喘模型组、哮喘模型延续组、组胺组、组胺受体拮抗剂组。用高效液相色谱法检测血清组胺浓度；生化分析仪测血清Na+、Cl-浓度；血气分析仪测血pH、PaO2、PaCO2、AB、SB；图像分析系统测定气道黏膜层、平滑肌层厚度。结果:（1）哮喘模型组血清组胺浓度、气道壁黏膜层和平滑肌层厚度均明显高于正常对照组 （P<0.01）；哮喘模型延续组明显高于哮喘模型组（P<0.01）；组胺组明显高于哮喘模型延续组（P<0.01）；而组胺受体拮抗剂组低于哮喘模型延续组（P<0.05，P<0.01）；（2）哮喘模型组的PaO2低于正常对照组（P<0.01）；哮喘模型延续组的PaO2、pH、AB、SB低于、PaCO2高于哮喘模型组（P<0.01）；组胺组PaO2、pH、AB、SB低于、PaCO2高于哮喘模型延续组（P<0.01）；而组胺受体拮抗剂组PaO2、pH、AB、SB高于哮喘模型延续组（P<0.01），PaCO2则低于哮喘模型延续组（P<0.01）。示豚鼠哮喘时存在气道重塑和血清组胺浓度升高以及代谢性酸中毒与呼吸性酸中毒，外源性组胺可加重这些变化，组胺受体拮抗剂可缓解之。结论:组胺在哮喘气道重塑中起介导作用，组胺受体拮抗剂对防治哮喘气道重塑及酸碱平衡紊乱有一定作用。     

口服白三烯受体拮抗剂与吸入糖皮质激素治疗小儿咳嗽变异型哮喘合并变应性鼻炎的对比研究

目的对比研究口服白三烯受体拮抗剂（LTRA）与吸入糖皮质激素（ICS）对dxJL咳嗽变异型哮喘合并变应性鼻炎的防治效果。方法54例2-5岁患儿随机分为LTRA组（27例）和ICS组（27例）。LTRA组口服孟鲁司特钠，每次4mg，睡前服用1次；ICS组患儿规律吸入布地奈德气雾剂，每日200-400μg，采用储雾罐辅助吸入。药物治疗3个月后继续随访观察15个月。结果LTRA组与ICS组比较．控制症状所需的平均天数为（8．31±3．69）d和（7．20±2．78）d；症状缓解率为92．6％和96．3％，差异无统计学意义（P〉0．05）。在病程的18个月LTRA组复发率（36．0％）显著高于ICM组（15．4％）。转化为典型哮喘者。LTRA组7例（28．0％），ICS组2例（7．7％），差异有统计学意义（P〈0．05）。结论口服LTRA控制小儿咳嗽变异型哮喘合并变应性鼻炎的近期疗效与ICS相当。可作为一线药物使用。但接受LTRA治疗的患儿远期复发率或者转化为典型哮喘的比率高于ICS治疗的患儿。     

白三烯受体拮抗剂介导NLRP3/Caspase-1/IL-1β信号通路改善支原体肺炎小鼠Th1/Th2免疫失衡

目的 探究白三烯受体拮抗剂对支原体肺炎小鼠Th1/Th2免疫的影响及可能机制。方法 30只BALB/c小鼠随机分为对照组（Control组）、肺炎支原体感染组（MP组）和白三烯受体拮抗剂孟鲁斯特（MK组）,通过滴鼻接种菌液建立支原体肺炎小鼠模型。取材后对各组小鼠计算肺湿重指数;HE染色观察肺组织病理变化;ELISA法检测肺泡灌洗液中IL-1β、IL-12、IFN-γ、TNF-α含量;TUNEL法检测肺组织细胞凋亡情况;流式技术检测外周血CD4+/CD8+水平;Western blot法检测肺组织NLRP3、pro-caspase1、pro-IL1β的表达水平。结果 白三烯受体拮抗剂改善支原体肺炎小鼠肺脏损伤,抑制IL-1β、IL-12、IFN-γ、TNF-α的分泌,抑制细胞凋亡,改善Th1/Th2免疫失衡,下调NLRP3、pro-caspase1、pro-IL1β的表达。结论 白三烯受体拮抗剂通过介导NLRP3/Caspase-1/IL-1β信号通路改善Th1/Th2免疫失衡,降低肺脏损伤。     

Th17淋巴细胞及相关细胞因子加重哮喘小鼠气道炎症

目的:研究Th17淋巴细胞及其相关的细胞因子在加重哮喘小鼠气道炎症中的作用机制.方法:20只小鼠随机均分为哮喘组和正常对照组.哮喘组用卵白蛋白(OVA)致敏与激发建立小鼠哮喘模型.对照组致敏与激发均以生理盐水代替.HE染色观察小鼠气道及肺组织病理变化;光学显微镜下观察小鼠支气管肺泡灌洗液(BALF)中细胞分类及计数;酶联免疫吸附试验(ELISA)检测小鼠BALF上清中IL-4、IFN-γ及IL-17的含量,流式细胞技术(FCM)检测小鼠外周血Th1、Th2及Th17淋巴细胞占CD4+细胞百分率情况.将外周血各T淋巴细胞亚群与BALF的中性粒细胞数作相关性分析.结果:哮喘组小鼠BALF中细胞数及中性粒细胞、嗜酸性粒细胞、淋巴细胞百分率均显著高于对照组(P＜0.05),BALF上清中IL-4和IL-17的水平显著增高(P＜0.05),而IFN-γ的水平显著降低(P＜0.05),外周血Th2、Th17淋巴细胞占CD4+细胞百分比显著增高(P＜0.05),而Th1淋巴细胞占CD4+细胞百分比显著降低(P＜0.05).相关性分析显示Th1淋巴细胞与BALF的中性粒细胞数呈显著负相关(rThl=-0.446,P＜0.05),Th17淋巴细胞与BALF的中性粒细胞数呈显著正相关(rTh17=0.394,P＜0.05).结论:Th17淋巴细胞可促进中性粒细胞及炎性介质在哮喘小鼠气道内的聚集,加重气道炎症.     

Costimulatory molecule OX40L is critical for both Th1 and Th2 responses in allergic inflammation

T cell activation and cytokine secretion are important mediators of inflammation in allergic asthma. The costimulatory pathway CD28/CD80/CD86 has been shown to play an important role in T cell activation in allergic asthma, but less is known about the effect of other costimulatory molecules in allergy. The costimulatory molecule OX40 ligand (OX40L), a member of the tumor necrosis factor superfamily, has been shown to be important in T cell priming and cytokine production. We investigated the role of OX40L in a murine model of allergic inflammation using OX40L(-/-) mice. In this model, following OVA sensitization and challenge, mice develop features of allergic inflammation including elevated levels of total serum IgE, pulmonary eosinophils, cytokines, and pulmonary inflammation. In the absence of OX40L, total serum IgE, pulmonary eosinophils, cytokines, and pulmonary inflammation were all significantly reduced compared to wild-type controls. Levels of eotaxin mRNA, an eosinophil-specific chemoattractant, were also markedly reduced, paralleling the significant reduction in pulmonary eosinophils. Levels of allergen-induced Th1 as well as Th2 cytokines were also significantly reduced. Together, the data support a critical role for OX40L signals in allergic responses.     

薯蓣皂苷减轻卵清蛋白诱导的过敏性哮喘小鼠的气道炎症

目的探讨薯蓣皂苷对卵清蛋白(ovalbumin,OVA)诱导的过敏性哮喘小鼠中过敏性支气管炎的影响及机制。方法24只小鼠随机分为对照组、OVA组、OVA+30 mg/kg薯蓣皂苷组和OVA+60 mg/kg薯蓣皂苷组,每组纳入6只小鼠。全自动生化仪检测各组支气管肺泡灌洗液(bronchoalveolar lavage fluid,BALF)中嗜中性粒细胞、嗜酸性粒细胞和单核细胞的数量;ELISA法检测BALF中促炎因子IL-1β、IL-4、IL-5和TNF-α的含量;PAS染色法观察肺组织黏液分泌情况并对黏液分泌程度进行评分;免疫组化法观察肺组织p-NF-κB p65的表达和分布情况;Western blotting法检测肺组织中p-IκB和NF-κB p65的蛋白表达水平。结果薯蓣皂苷可降低过敏性哮喘小鼠BALF中炎性细胞数量,同时降低BALF中促炎因子IL-1β、IL-4、IL-5和TNF-α的水平以及肺部黏液的分泌和NF-κB的活化水平。结论薯蓣皂苷能减轻过敏性哮喘小鼠的气道炎症,且其抗炎作用与抑制NF-κB活化有关。     

Th17 responses in chronic allergic airway inflammation abrogate regulatory T-cell-mediated tolerance and contribute to airway remodeling

The role of T-helper type 17 (Th17) responses in airway remodeling in asthma is currently unknown. We demonstrate that both parenteral and mucosal allergen sensitization, followed by allergen inhalation, leads to Th17-biased lung immune responses. Unlike Th17 cells generated in vitro, lung Th17 cells did not produce tumor necrosis factor-α or interleukin (IL)-22. Eosinophilia predominated in acute inflammation, while neutrophilia and IL-17 increased in chronic disease. Allergen-induced tolerance involved Foxp3-, Helios-, and glycoprotein-A repetitions predominant-expressing regulatory T cells (Treg) and IL-10/interferon-γ priming. This Treg phenotype was altered in inflamed lungs and abrogated by inhalation of IL-17. Using Th17-deficient mice with genetic disruption of gp130 in T cells, we showed that Th17 cells induce airway remodeling independent of the Th2 response. All-trans retinoic acid administration ameliorated Th17-mediated disease and increased Treg activity, while dexamethasone inhibited eosinophilia but not neutrophilia, and enhanced Th17 development in vitro. Targeting the Th17/Treg axis might therefore be therapeutic in neutrophilic and glucocorticoid-refractory asthma.     

Creatine supplementation exacerbates allergic lung inflammation and airway remodeling in mice

Creatine supplement is the most popular nutritional supplement, and has various metabolic functions and sports medicine applications. Creatine supplementation increases muscle mass and can decrease muscular inflammation. Some studies have also suggested a beneficial role of creatine supplementation on chronic pulmonary diseases such as chronic obstructive pulmonary disease and cystic fibrosis. Among athletes, the prevalence of asthma is high, and many of these individuals may be taking creatine. However, the effects of creatine supplementation on chronic pulmonary diseases of allergic origin have not been investigated. In the present study, we analyzed the effects of creatine supplementation on a model of chronic allergic lung inflammation. Thirty-one Balb/c mice were divided into four groups: control, creatine (Cr), ovalbumin (OVA), and OVA+Cr. OVA and OVA+Cr groups were sensitized with intraperitoneal injections of OVA on Days 0, 14, 28, and 42. OVA challenge (OVA 1%) and Cr treatment (0.5 g/kg/d) were initiated on Day 21 and lasted until Day 53. We determined the index of hyperresponsiveness, the serum levels of OVA-specific immunoglobulin (Ig)E and IgG(1), and the total and differential cell counts in bronchoalveolar lavage fluid. We also quantified airway inflammation, and the airway density of IL-4+, IL-5+, IL-2+, IFN-gamma+, and insulin-like growth factor (IGF)-1+ cells, collagen and elastic fibers, and airway smooth muscle thickness. Our results showed that creatine in OVA-sensitized mice increased hyperresponsiveness; eosinophilic inflammation; airway density of IL-4+, IL-5+, and IGF-1 inflammatory cells; airway collagen and elastin content; and smooth muscle thickness. The results show that creatine supplementation exacerbates the lung allergic response to OVA through a T helper cell type 2 pathway and increased IGF-1 expression.     

二甲双胍抑制慢性哮喘小鼠气道炎症、重塑及新生血管形成

目的:探讨二甲双胍对慢性哮喘气道炎症、重塑及新生血管形成的影响及可能机制。方法:采用卵白蛋白(OVA)致敏并激发制备慢性哮喘小鼠模型,给予二甲双胍干预,与生理盐水对照组和慢性哮喘模型组相比,观察支气管肺泡灌洗液(BALF)细胞计数、外周血免疫球蛋白、气道重塑及磷酸化腺苷酸活化蛋白激酶(pAMPK)蛋白水平的变化。结果:慢性哮喘小鼠BALF细胞总数及嗜酸性粒细胞百分比较对照组升高(P0.01),血清OVA特异性IgE明显升高(P0.01),给药组可降低上述指标(P0.05)。肺组织HE染色可见气道壁炎症细胞浸润、杯状细胞增生、上皮下胶原沉积等病理改变,免疫组化CD31染色观察到气道上皮下新生血管数目和面积增加;二甲双胍部分抑制了上述病理过程。肺组织免疫组化p-AMPK染色观察到其在模型组气道壁的表达较对照组下降(P0.05),给药组升高明显(P0.01)。结论:慢性哮喘中AMPK磷酸化表达水平受抑制。二甲双胍可能通过激活AMPK来抑制慢性哮喘气道炎症、重塑及新生血管的形成。     

The effect of allergen-induced airway inflammation on airway remodeling in a murine model of allergic asthma

OBJECTIVE AND DESIGN: We examined the effect of airway inflammation on airway remodeling and bronchial responsiveness in a mouse model of allergic asthma. MATERIALS AND METHODS: BALB/c mice were sensitized to ovalbumin (OA), and exposed to aerosolized OA (0.01, 0.1 and 1%). Twenty-four hours after the final antigen challenge, bronchial responsiveness was measured, and bronchoalveolar lavage (BAL) and histological examinations were carried out. RESULTS: Repeated antigen exposure induced airway inflammation, IgE/IgG1 responses, epithelial changes, collagen deposition in the lungs, subepithelial fibrosis associated with increases in the amount of transforming growth factor (TGF)-beta1 in BAL fluid (BALF), and bronchial hyperresponsiveness to acetylcholine. The number of eosinophils in BALF was significantly correlated with TGF-beta1 production in BALF and the amount of hydroxyproline. Furthermore, significant correlations were found between these fibrogenic parameters and the bronchial responsiveness. CONCLUSION: These findings demonstrated that in this murine model airway eosinophilic inflammation is responsible for the development of airway remodeling as well as bronchial hyperresponsiveness in allergic bronchial asthma.