【作者】 黄华琼；

【导师】 沈华浩；

【作者基本信息】 浙江大学 ， 内科学， 2008， 博士

【摘要】 研究背景支气管哮喘(简称哮喘)是威胁人类健康的最常见呼吸疾病之一,其发病率在全世界范围内有增高趋势。全球有超过3亿哮喘患者,我国约有3900万患者。目前以糖皮质激素为主的哮喘综合治疗,虽然能使哮喘患者得到良好的控制,但激素长期使用引起的不良反应,疾病带来的患者精神和经济问题,不仅导致患者生活质量下降,也给社会造成重大损失。迄今为止,哮喘尚无有效的预防手段。卡介苗(BCG)是结核分枝杆菌减毒活疫苗,具备免疫调节作用,能诱导机体产生强烈的Th1反应,1997年Shirakawa等在《Science》杂志报道,在日本的学龄儿童中,结核菌素的迟发相超敏反应强度与特异质呈负相关。该发现为BCG应用于哮喘预防带来了曙光。BCG作为强烈的Th1型细胞因子诱导剂,在Th2细胞因子占主导地位的哮喘的预防作用备受关注。来自动物实验的结果几乎无一例外的支持BCG对变态反应性疾病的预防作用。动物实验研究显示BCG接种能明显抑制OVA致敏和激发所致的气道嗜酸性粒细胞浸润,降低气道高反应性以及Th2型细胞因子的表达等。在人群流调方面,从1997年日本学者发现BCG接种和哮喘发病呈负相关的关系后,在几内亚、巴西、匈牙利以及中国北京等多个国家和地区进行的调查发现结核菌素皮试阳性与变态反应性疾病的发展呈负相关,提示BCG接种可能是有效的变态反应性疾病的预防途径。国外临床实验研究显示,轻中度哮喘患者接种BCG后FEV1、mPEFR均较对照组明显改善,结核菌素试验反应增强;在诱导痰中嗜酸性粒细胞数量较对照组低,而IFN-γ/IL-4比值增高。一年后再次皮下接种BCG,发现重复BCG接种能更好改善肺功能,进一步提高血清IFN-γ/IL-4比值。然而另有其他研究认为BCG接种与哮喘发病无相关性。我们已在国内外率先观察了新生幼鼠早期多次BCG接种对哮喘气道炎症和气道黏液分泌的影响,并得到了令人振奋的结果:早期多次BCG接种可抑制哮喘小鼠气道炎症,降低气道黏液高分泌。我们随后的研究还发现,早期多次接种BCG对小鼠哮喘的预防具有长期的保护作用,长达45周之久。我们将在上述研究基础上,通过与新生鼠单次接种的对比,进一步研究早期多次接种对诱导机体Th1/Th2免疫的作用以及对成年小鼠哮喘的预防作用。结核(TB)是呼吸系统疾病中的另一健康杀手,全球每年有超过8百万新发病例,2-3百万死亡病例。随着移民潮的涌现、HIV人群增加以及结核菌耐药菌株的出现,全球结核疫情处于非常危险的境地。BCG因其安全、廉价、制备方便已在全球使用了80多年,然而对TB的保护作用却颇有争议。世界各地的研究结果显示BCG对TB的保护效率从0-80%不等,Graham等进行的一项荟萃分析认为BCG对TB的保护效率为50%。因此研究如何提高BCG对TB的保护效率,探寻新的预防策略已成为迫在眉睫的任务。复种策略一直是研究如何提高BCG预防结核效率所关注的内容。在人群中的调查显示复种的效果不一致。在匈牙利、波兰等国家进行的研究认为复种有效,而非洲Malawi地区进行的研究调查显示复种无益于提高BCG的保护作用。并且有研究发现成年后复种产生的保护作用维持的时间很短。为什么会出现不同的复种效果?在进一步的动物研究中发现,环境因素,如环境分枝杆菌暴露或寄生虫感染,以及初种和复种之间的时间间隔是影响BCG保护作用的重要因素。环境分枝杆菌暴露或寄生虫感染可能降低BCG预防结核的作用。另外有研究指出恰当的初种和复种间隔可能提高和延长BCG的保护作用。J.F.T.Griffina等进行的研究两次接种时间最短间隔为4周,最长为43周,结果发现间隔时间过长反而抑制BCG的保护作用。因此有必要在生命早期即开展多次接种,并缩短复种之间的间隔,以探索早期短间隔复种BCG预防结核的作用。在本研究中,采用新生鼠早期、多次、短间隔BCG接种的策略,观察该接种方法对生命后期哮喘以及结核的保护作用,从而为BCG作为哮喘疫苗提供实验依据,为提高人类结核疫苗的接种效率提供新策略。第一部分早期多次接种BCG预防哮喘目的:新生鼠多次接种BCG,待成年后诱发哮喘,观察早期接种卡介苗对诱导机体Th1、Th2免疫的作用,以及对致敏原激发后气道反应性、嗜酸粒细胞浸润为主的气道炎症以及黏液高分泌的作用;方法:选择C57BL/6新生鼠,随机分为早期多次BCG组(BCG3组)、早期单次BCG组(BCG1组)和对照组(saline组)。BCG3组分别在出生0、7、14天予以10~5CFU BCG行皮下接种3次,BCG1组仅在出生0天予10~5CFU BCG接种1次,对照组给予等量注射用水。待小鼠8周龄后检测各组脾细胞上清IFN-γ、IL-4水平。随后以卵白蛋白(ovablumin,OVA)进行致敏和激发,建立哮喘小鼠模型。各组小鼠(BCG3+OVA组,BCG1+OVA组,OVA组,saline组)于最后一次抗原激发后48小时检测气道反应性、进行支气管肺泡灌洗液细胞分类计数、肺组织病理切片观察气道炎症和黏液高分泌。结果:经BCG接种后,8周龄接种小鼠脾上清IFN-γ高于未接种组。与BCG1组比较,BCG3组IFN-γ水平显著升高,有统计学意义(P＜0.05)。BCG3组和BCG1组脾上清IL-4水平与未接种组比较,无明显差别(P＞0.05)。各组小鼠经OVA激发后,OVA组小鼠气道反应性、支气管肺泡灌洗液中Eos、肺组织炎症和气道黏液分泌较生理盐水对照组明显增加(P＜0.05)。BCG1+OVA组与OVA组相比无显著差异(P＞0.05)。BCG3+OVA组气道高反应性较模型组明显减轻(P＜0.05)。BCG3+OVA组小鼠未见抑制致敏小鼠抗原攻击后引起的BALF细胞总数的增多,但显著降低了Eos数(P＜0.05)。病理学检查结果显示BCG3+OVA组肺组织炎症浸润和黏液高分泌明显降低(P＜0.05)。结论:新生鼠BCG接种可以诱导机体产生IFN-γ,并维持至成年后。新生鼠多次BCG接种诱导的Th1反应强于新生鼠单次BCG接种。经抗原致敏和激发后,新生鼠多次BCG接种明显抑制成年鼠OVA激发后的AHR、气道Eos炎症及黏液高分泌,而单次BCG接种未能抑制。早期多次BCG接种预防哮喘的机制可能依赖诱导产生的Th1型细胞因子。第二部分早期多次接种BCG预防结核目的:新生鼠多次接种BCG,待成年后予H37Rv攻击建立结核模型,观察:①早期接种BCG对结核模型小鼠一般情况、脾指数、肺和脾组织结核菌增殖、肺病理变化的作用:②Th1和Th2型细胞因子在结核免疫中的作用。方法:选择C57BL/6新生鼠,随机分为早期多次BCG组(BCG3组)、早期单次BCG组(BCG1组)和对照组(saline组)。BCG3组分别在出生0、7、14天予以10~5CFU BCG行皮下接种3次,BCG1组仅在出生0天予10~5CFU BCG接种1次,对照组给予等量注射用水。H37Rv攻击前,检测各组脾细胞上清IFN-γ、IL-4水平。待小鼠满8周龄后以2.5×10~6CFU H37Rv予尾静脉注射,建立结核小鼠模型。观察各组小鼠(BCG3+H37Rv组,BCG1+H37Rv组,H37Rv组,saline组)一般情况和生存情况,并选择在H37Rv攻击后3周、6周和9周收集标本,分别检测脾指数、肺和肝组织结核菌增殖和肺组织病理变化情况。同时检测这三个时间点小鼠脾细胞上清IFN-γ、IL-4水平和分泌IFN-γ的淋巴细胞频率,并进一步检测外周血IFN-γ、IL-12、IL-2和TNF-α水平。结果:各组小鼠经H37Rv攻击后3周,与H37Rv组小鼠比较,两组BCG组均显示对结核有抑制作用。与H37Rv组比较,BCG3+H37Rv组中脾指数显著降低(P＜0.05),而BCG1+H37Rv组脾指数无显著下降(P＞0.05)。BCG3+H37Rv组和BCG1+H37Rv组均显著降低肺、肝的结核菌负荷量和肺组织病变,其中,BCG3+H37Rv组肺组织中结核菌负荷量和肺组织病变严重程度均较BCG1+H37Rv组低,两组比较有统计学差别(P＜0.05)。至H37Rv攻击后6周,2组BCG干预组脾指数与H37Rv组比较无显著性差别(P＞0.05)。两组BCG组均仍显示肺、肝结核菌负荷明显低于H37Rv组(P＜0.05)。但BCG3+H37Rv组和BCG1+H37Rv组比较无统计学差别(P＞0.05)。肺组织病理显示2组BCG干预组病变进展,但累及范围仍明显小于H37Rv组。两组BCG干预组相互比较无明显差别。至H37Rv攻击后9周,2组BCG组脾指数、肺、肝结核菌负荷均上升,与H37Rv组比较无差别(P＞0.05)。肺组织病理显示2组BCG干预组病变范围与H37Rv组比较无差别。H37Rv感染前,8周龄BCG接种小鼠脾上清IFN-γ高于未接种组。与BCG1组比较,BCG3组IFN-γ水平显著升高,有统计学意义(P＜0.05)。感染后3周、6周、9周,H37Rv组、BCG3+H37Rv组和BCG1+H37Rv组IFN-γ水平均显著升高,与saline组比较有统计学差别(P＜0.05)。各时间点H37Rv组、BCG3+H37Rv组和BCG1+H37Rv组相互比较无统计学差别(P＞0.05)。进一步检测分泌IFN-γ的淋巴细胞频率发现,H37Rv感染的各组分泌IFN-γ的淋巴细胞频率增高,与saline组比较有统计学差别(P＜0.05)。仅在6周时,BCG3+H37Rv组与H37Rv组比较,分泌IFN-γ的淋巴细胞频率显著增高(P＜0.05)。H37Rv感染后3周,H37Rv组TNF-α水平较saline组明显升高(P＜0.05)。两组BCG干预组TNF-α水平被显著抑制(P＜0.05)。BCG3+H37Rv组和BCG1+H37Rv组之间无明显差异。至感染后6周和9周,随着结核模型组TNF-α下降,两组BCG组与结核组比较,TNF-α水平无明显显著差别(P＞0.01)。IL-2水平不受H37Rv感染影响,各组各时间点相互比较无显著差异(P＞0.05)。H37RV感染使IL-12水平升高,但BCG3+H37Rv组和BCG1+H37Rv组与相应时间点H37Rv组比较无明显差异(P＞0.05)。H37Rv感染前,BCG接种对小鼠脾上清IL-4水平无明显影响。H37Rv感染后3周、6周、9周等3个时间点动态监测脾细胞上清IL-4水平发现,感染后3周,经H37Rv攻击的各组小鼠,H37Rv组、BCG3+H37Rv组、BCG1+H37Rv组IL-4水平均显著升高(P＜0.05),与Saline组比较有统计学差别(P＜0.05)。与H37Rv组比较,2组BCG干预组IL-4明显降低(P＜0.05),但BCG3+H37Rv组和BCG1+H37Rv组相互比较无显著差别。感染后6周,经H37Rv攻击的各组小鼠IL-4水平继续保持升高,H37Rv组、BCG3+H37Rv组和BCG1+H37Rv组3组之间比较没有差别(P＞0.05)。感染后9周,H37Rv组IL-4明显回落。与H37Rv组比较,BCG3+H37Rv组和BCG1+H37Rv组IL-4呈显著升高(P＜0.05)。结论:新生鼠多次BCG接种预防结核作用仅在感染早期明显优于单次BCG接种。感染后期,新生鼠多次BCG接种和BCG单次接种出现预防作用逐渐下降,病变进展。这一动态变化可能与BCG在感染早期抑制TNF-α水平有关。Th1细胞因子不能作为机体具有结核保护力的指标,Th2细胞因子提示结核病病变处于进展期,并诱导TNF-α对机体的毒性作用。更多还原

【Abstract】 BackgroundAsthma is a chronic inflammatory disease characterized by airway infiltration of inflammatory cells,particularly eosinophils and T lymphocytes,mucous over-production,and airway hyperresponsiveness(AHR).The incidence and severity of asthma are increasing in the world and asthma is estimated to affect about 300 million persons.Currently,many anti-asthmatic medicines,including inhale corticosteroids,are available for controlling asthmatic attack.However,those treatments are directed at reducing symptoms and have little effect on inhibiting the disease progression.Therefore,preventative interventions may be a better approach to protect from asthma.Mycobacterium boris Bacillus Calmette-Guerin(BCG)is a strong inducer of the Th1 type immune response.At 1997,Shirakawa and his colleagues claimed that there was inverse relationship between tuberculin responses and atopic disorder,which cost light on the develepment of vaccine on asthma.BCG was viewed to induce Th1 type response and suppress Th2-mediated response in asthmatic model.Several studies have demonstrated that BCG can inhibit airway eosinophilia and decrease-related mucous over-production,reducing AHR in animal models of asthma.However,the relationship between BCG infection and asthma in human being is controversial.At 1997,Shirakawa and his colleagues claimed that there was inverse relationship between tuberculin responses and atopic disorder,some following studies came from Guinea,Brazil,Hungary and China also found that BCG vaccination was associated with lower asthma prevalence.A recent clinical study showed repeated BCG further improved the lung function and increased the IFN-γ/IL-4 ratio in the peripheral blood in mild to moderate asthmatic patients.But a large body of data suggested there was no protective effect of BCG vaccination against atopy.And our laboratory has demonstrated that early bacillus calmette-guerin vaccination prevents airway mucus production in mouse models of asthrna. Besides,our recent work investigates the protection induced by multiple BCG vaccination at newborn on the airway inflammation of asthmatic mice at different age. We found neonatal multiple BCG vaccination elicited long-term protection in mouse allergic responses,even at 45 week old mice.To further investigate the effect of neonatal multiple BCG vaccination on adult allergic symptoms,we tried to find the difference level of protection and T cell response induce by multiple BCG or single BCG vaccination at newborn.Tuberculosis is a global health problem and it is estimated roughly that one third of the world’s population is infected with Mytobactium tuberculosis and approximately 8-10 million people become infected each year.With people’s move frequently,the spread of HIV/AIDS infection and the emergence of multi-drug-resistant strains of M. tubercolusis,the risk of tubercolusis is increased.Till now,M.bovis BCG is the only available anti-tubercolusis vaccine in the world wide.However,its role in adult has been highly variable from 0-80%.A META data showed that the efficacy of BCG on tuberculosis is about 50%.The development of an improved efficacy of vaccine or new vaccination remege against tuberculosis is urgently required and the identification of immune response correlates for protection would greatly facilitate the rational design of such a vaccine.Given the safety and very extensive coverage of BCG,repeated BCG vaccination might be a clinical strategy.However,revaccination of humans with BCG is controversial,some studies had demonstrated that repeated BCG concluded that revaccination was useful,others denied the increasing protective efficacy of the primary vaccination.In addition,the protection induced by BCG was reported to last for short term.Besides,the factors which impact on the efficacy of revaccination or the reasons why revacciantion failed to protect TB were known little. Evidence from animal study indicated that the environmental situation,such as the exposure of environmental mytobacteria,warm infection would deduce the protection efficacy of BCG on tuberculosis.Becides,it was reported that the internal of primary and boost vaccination played a privotal role on the protection.The authors suggested that long internal,of vaccination reduced the protection induced by multiple BCG vaccination.We hyposisth that neonatanl mice was vaccinated and boosted with BCG at a short internal would enhance the efficacy of BCG against TB.Part 1 Neonatal multiple Bacillus Calmette-Guerin elicits a superior protection in mouse allergic responsesObjectiveTo investigate whether revaccination of neonatal mice with BCG increases the level of protection against allergic responses induced by a single BCG vaccination.MethodsC57BL/6 neonates were vaccinated with BCG for three times on days 1,7 and 14 or only vaccinated on day 1.When part of these mice grew up to 8 weeks old,IFN-γand IL-4 level were tested in splenocyte supernatants by ELISA.And others were sensitized with ovalbumin at 8 and 10 weeks of age,then challenged with allergen at 12 weeks of age.Their airway inflammation was characterized.Results8 weeks later,IFN-γwere high in BCG immunized mice,and multiple BCG vaccination increased IFN-γlevel significantly as compared with single BCG immunization(P＜0.05).IL-4 response was similar in mice with BCG immunized or without.After OVA sensitization and challenge,only multiple BCG vaccination inhibited airway hyperresponsiveness,eosinophils infiltration and mucus overproduction(P＜0.05),while single BCG vaccination on neonates had little effect on allergic responses.ConclusionOur data suggest that neonatal BCG vaccination induced predominated IFN-γresponse,which lasted to adult lifetime.And multiple BCG elicits robust IFN-γresponse.Also,multiple BCG immunization has an effect on inhibiting airway hyperresponsiveness,eosinophilia and mucus overproduction.Single BCG vaccination at newborns had no protection on the allergic response.Thus,neonatal multiple BCG vaccination has a protective effect on adult mice,which may be mediated by IFN-γresponse.Part2 Repeated Bacillus Calmette-Guérin vaccination at newborns elicits a superior but short-term protection in M.tuberculosis infected mouseObjectivesTo investigate whether revaccination of neonatal mice with BCG increases the level of protection against tuberculosis induced by a single BCG vaccination.MethodsC57BL/6 neonates were vaccinated with BCG for three times on days 1,7 and 14 or only vaccinated on day 1.When part of these mice grew up to 8 weeks old,IFN-γand IL-4 level were tested in splenocyte supernatants.And other parts of mice were challenged with M.tuberculosis H37Rv.At 3,6 and 9 weeks of post challenge,their tuberculosis burden in organs and histologic change in lung,as well as IFN-γ,IL-4 level in splenocyte supematants,frequency of IFN-γproducing cells of spleen and IFN-γ,IL-2,IL-12 and TNF-αin serum were characterized. ResultsAt 3 weeks of M.tuberculosis H37Rv challenge,BCG vaccination inhibited tuberculosis growth in lung and liver significantly(P＜0.05)and reduced lung tissue damage.Multiple BCG vaccinated mice gained lower spleen/body weight and M. tuberculosis burden as well as mild lung histological damaged in lung than those in single BCG immunized mice(P＜0.05).At week 6,however,a time-related decline of effect was appeared in both multiple and single BCG vaccinated mice.The area of lung tissue damage was enlarging and the replication of M.tuberculosis was progressing,although which were significantly different from tuberculosis mice.Till to 9 weeks of challenge,there has no any significant difference in burden of M. tuberculosis or histological change between multiple BCG vaccinated,single BCG vaccinated and infected but non-vaccinated mice.Th1 cytokines were associated with M.tuberculosis infection or BCG immunization. 8 weeks post BCG immunization,IFN-γwere upregulated,and multiple BCG vaccination increased IFN-γlevel significantly as compared to single BCG immunization(P＜0.05).3,6,9 weeks post of infection,IFN-γlevel was significantly increased in mice even infected by M.tuberculosis,compared to non-infected mice. There was no significant difference in mice with BCG immunized or without.To further assess the association of IFN-γresponse and protection against tuberculosis, the frequency IFN-γ-producing cell of spleen was detected.The data showed that there was no relationship with BCG vaccination-induced IFN-γproducing cell and protection against tuberculosis in 3 weeks of infection.Other Thl cytokines,IL-2 and IL-12 in serum didn’t be affected by repeated or single BCG immunization,but the level of TNF-αwas inhibited significantly in two BCG groups at 3 weeks of infection (P＜0.05),while multiple and single BCG vaccinated mice had no significant difference in TNF-αlevel.IL-4 responses were similar in mice with BCG immunized or without before M. tuberculosis infection.At 3 weeks of infection,Th2-type cytokine in vitro were significantly up-regulated in HRV37 challenged mice as compared to saline group(P＜0.05).Th2 response was significantly lower in BCG immunized mice than in M. tuberculosis infected mice(P＜0.05).Till to 9 week of challenge,as disease processing in those BCG immunized mice and limiting in M.tuberculosis mice,IL-4 level reversed to significant high in BCG immunized mice when compared to M. tuberculosis infected mice(P＜0.05).There was no significant difference between multiple and single BCG vaccinated mice at every time point.ConclusionOur data suggest that revaccination of neonatal mice with BCG has a superior but short-term effect on tuberculosis.The time-related decline of protection may be induced by restrained TNF-αlevel induced by BCG.And the correlation between Th1 response and protection against tuberculosis was poor.Th2 response indicated to involve in the progression of tuberculosis and induced the toxic of TNF-α.更多还原