BN大鼠致敏动物模型研究

目的建立经腹腔注射途经给予造模致敏原的BN大鼠致敏动物模型。方法分别在实验第1、5和10天,经腹腔注射给予不同性别(雌性和雄性)和不同周龄(4周龄和8周龄)的BN大鼠,不同剂量(1.00、0.10和0.01mg)的造模致敏原———卵清蛋白(OVA),观察35天。分别于第28、35天内眦取血分离血清,用ELISA方法检测血清中OVA特异性IgE。结果与阴性对照组相比,雌性8周龄BN大鼠中、高剂量组第28、35天血清中OVA特异性IgE浓度显著升高(P<0.05);雄性8周龄BN大鼠低、中剂量组血清中OVA特异性IgE浓度在第28天显著升高(P<0.05),高剂量组血清中OVA特异性IgE浓度在第35天显著升高(P<0.05);雄性4周龄BN大鼠,高剂量组血清中OVA特异性IgE浓度在第35天显著升高(P<0.05)。结论经腹腔注射途径给予不同性别和周龄的BN大鼠不同剂量的OVA,雌性大鼠比雄性大鼠更敏感;8周龄大鼠比4周龄大鼠更敏感;0.10mg或1.00mg的致敏剂量较适合。因此,选用雌性8周龄BN大鼠,经腹腔注射给予0.10mg或1.00mgOVA,35天后即可建立比较理想的BN大鼠致敏动物模型。     

小鼠过敏性肠炎模型的免疫学研究

目的 评价卵清蛋白(OVA)饲喂致敏OVA特异性T细胞受体转基因小鼠(OVA23-3)的方法建立过敏性肠炎模型的可行性,并观察其免疫学指标变化.方法 采用高OVA饲料饲喂OVA23-3小鼠的方法,建立小鼠过敏性肠炎模型.通过ELISA法检测血清OVA特异性IgE抗体和细胞培养上清细胞因子的含量,流式细胞术检测细胞内细胞因子.结果 高OVA饲料饲喂引起OVA23-3小鼠体重下降,并发生以小肠为主的出血性炎症.与对照组比较,实验组血清OVA特异性IgE含量明显增高,细胞培养上清IL-4水平增高,而IFN-β水平降低,同时CD4+T细胞中IL-4产生细胞百分率显著增加,IFN-γ产生细胞百分率显著减少.结论 高OVA饲料饲喂OVA23-3小鼠诱发过敏性肠炎的方法是一种理想快速简便的方法,Th2型细胞反应占优势是其重要特征.     

经口致敏食物过敏大鼠模型的建立

目的探讨经口致敏食物过敏大鼠模型的建立方法,以及适宜的评价指标。方法以卵清蛋白(OVA)作为致敏原,将16只3周龄Brown-Norway(BN)大鼠随机分为3组:阴性对照组(生理盐水灌胃)、阳性对照组(OVA腹腔注射组)和实验组(OVA灌胃组),共灌胃9周。在第4、5、6、7、8、9周分别采用双抗体夹心ELISA法及皮肤过敏实验法(PCA)测定实验动物血清OVA特异性IgE(OVA-IgE)抗体滴度,以判断动物是否致敏成功,在第13周各组动物给予100mg/mlOVA1ml灌胃激发后测血清OVA-IgE滴度。结果ELISA结果显示在灌胃第6周时实验组动物OVA-IgE滴度水平达到最高,且显著高于阴性对照组(P<0.05),致敏率为60%(3/5);第7、第8周时OVA-IgE滴度水平略有下降,但是仍然显著高于阴性对照组(P<0.05),致敏率达到80%(4/5),OVA-IgE水平和致敏率与阳性对照组之间差异无显著性;实验组PCA结果在第6、7、8周均显示为阴性,而阳性对照组PCA显示为阳性。结论经口致敏可作为一种可行的建立食物过敏动物模型的方法,符合食物过敏发生的自然生理过程,建议适宜致敏时间为6周;采用ELISA法检测血清OVA-IgE抗体水平较PCA法能更加灵敏地反映经口致敏模型是否成功。     

删除CD4~+CD25~+T细胞对OVA诱导的小鼠体液免疫应答的影响

目的观察删除CD4+CD25+T细胞对OVA免疫小鼠体液免疫应答的影响。方法小鼠腹腔注射抗CD25单克隆抗体,分别于3天、10天、27天采血流式检测外周血中CD4+CD25+T细胞的比例;注射抗CD25单克隆抗体3天后,OVA加铝佐剂免疫未删除和删除CD4+CD25+T细胞小鼠,7天后加强免疫一次,分别于初次免疫后7天,加强免疫后14天采血制备血清,ELISA法检测血清中OVA特异性IgE和IgG1的浓度。结果注射抗CD25单克隆抗体3天和10天,外周血中无CD4+CD25+T细胞;27天后CD4+CD25+T细胞的比例部分恢复。初次免疫7天后,删除CD4+CD25+T细胞小鼠总IgE、OVA特异性IgE和IgG1浓度较未删除组升高;加强免疫14天后,删除CD4+CD25+T细胞小鼠OVA特异性IgE较未删除组升高。结论删除CD4+CD25+T细胞能够影响小鼠OVA特异性体液免疫应答。     

鸡卵蛋白致敏小鼠TH1/TH2平衡的变化及其减毒活菌卡介苗干预的影响

目的研究鸡卵蛋白(OVA)致敏小鼠体内TH1/TH2平衡的变化,探讨减毒活菌卡介苗(BCG)对其的影响以及作用机制. 方法所有实验小鼠被分为OVA致敏组(组1)、OVA致敏+BCG干预组(组2)及正常对照小组(组3),每组8只.第一组小鼠于第0天给予OVA10mg腹腔注射致敏.第二组小鼠在给予OVA腹腔注射的同时皮下注射BCG干预.第三组小鼠在第0天腹腔注射生理盐水对照.全部实验小鼠于第14天处死,行支气管肺泡灌洗术(BAL),测定肺泡灌洗液(BALF)中的细胞总数及嗜酸性粒细胞(EOS)的个数.开腹取脾,制备脾单细胞悬液并培养48h,收集上清液.Elisa法测定上清液中的IL-4、IFN-γ的含量.结果致敏组小鼠BALF中的细胞总数较正常对照组增高,给予BCG干预后细胞总数与致敏组相比无明显变化.三组小鼠BALF中均未发现EOS. 与正常对照组相比,致敏组脾细胞培养上清液中的IL-4水平增高、IFN-γ水平降低.经BCG干预后能提升IFN-γ水平,同时降低IL-4水平.结论致敏小鼠体内TH2细胞功能亢进.BCG干预后可以上调TH1细胞应答.     

皮肤暴露邻苯二甲酸二丁酯对小鼠过敏性哮喘的影响

目的 探究皮肤暴露邻苯二甲酸二丁酯(Dibutyl phthalate,DBP)对过敏性哮喘的影响.方法 30只Balb/c雄性小鼠随机分为5组:(1)对照组;(2) Ovalbumin (OVA)致敏组;(3)4 mg/kg/d DBP+ OVA组;(4) 40 mg/kg/d DBP+ OVA组;(5)400 mg/kg/d DBP+ OVA组.第1～5组小鼠每天皮肤暴露于生理盐水或不同浓度的DBP,连续28 d.第2～5组小鼠分别在实验的11、18及25 d腹腔注射OVA致敏液,并且在第29～35 d进行每日一次每次30 min的OVA(1％)雾化,构建小鼠过敏性哮喘模型.第36 d处死实验小鼠,取肺组织和血清,使用ELISA法检测肺组织中的白介素-4(interleukin-4,IL-4)、干扰素-γ(interferon-γ,IFN-γ)和血清中的免疫球蛋白E(immunoglobulin E,IgE)含量.同时,采用HE染色法观察小鼠肺部气道的病理学变化.结果 与过敏性小鼠模型组相比,DBP皮肤暴露会提高肺组织中IL-4和血清中IgE的含量,具有统计学意义(P＜0.01).同时,会显著的加重小鼠肺组织的气道重塑和炎性细胞浸润等病理学变化.结论 长期皮肤暴露于DBP(＞4 mg/kg/d)会诱导小鼠产生过敏性体制,加重过敏原诱导过敏性哮喘.     

互隔交链孢霉诱导小鼠气道变态反应性炎症动物模型的构建

目的研究使用互隔交链孢霉孢子构建C57BL/6小鼠肺部变态反应性炎症模型的方法。方法模型组、阳性对照组和阴性对照组各12只小鼠。模型组以互隔交链孢霉孢子悬液致敏和激发小鼠,分别于第0、5、10天腹腔注射霉菌孢子悬液,第14天开始连续4天鼻滴霉菌孢子悬液。阳性对照组以鸡卵蛋白(OVA)代替霉菌孢子,阴性对照组以磷酸缓冲液(PBS)代替霉菌孢子。进行肺组织病理检查,支气管肺泡灌洗液(BALF)中细胞计数分类和总蛋白浓度测定,ELISA测定BALF中的白细胞介素-4(IL-4)和干扰素-γ(IFN-γ)及血浆中的总IgE和特异性IgE,测定小鼠气道阻力。结果模型组和阳性对照组可见明显炎症细胞浸润,特别是嗜酸性粒细胞,而阴性对照组则未见。BALF中细胞总数和嗜酸性粒细胞比例较阴性对照组明显升高(P<0.05);BALF上清中IL-4、总蛋白水平较阴性对照组明显升高(P<0.05),IFN-γ水平较阴性对照组降低(P<0.05)。血浆中总IgE和特异性IgE较阴性对照组明显升高(P<0.05)。小鼠气道阻力较阴性对照相比有明显升高(P<0.05)。结论使用互隔交链孢霉孢子致敏和激发C57BL/6小鼠成功的建立了小鼠气道变态反应性炎症模型。     

灭活卡介苗对哮喘小鼠治疗作用及机制探讨

目的 探讨灭活卡介苗治疗哮喘小鼠的作用机制.方法 40只Balb/c小鼠随机分4组,模型组:皮下注射卵蛋白(OVA)加氢氧化铝凝胶首次致敏,第7、14天用等量致敏液腹腔注射,第21天后用OVA超声雾化,连续激发7 d.对照组:用生理盐水代替OVA氢氧化铝混合液注射,雾化用生理盐水代替OVA,处理同模型组.治疗组:致敏激发同模型组,连续激发7 d之后,以灭活卡介苗皮内注射,1次/周,共13周,每隔1月用OVA超声雾化激发3 d.治疗对照组处理:激发同治疗组,用生理盐水代替灭活卡介苗皮内注射,时间同治疗组.所有动物均在末次激发后处死,分别行肺组织切片及支气管肺泡灌洗,取灌洗液行细胞分离与计数、细胞因子IL-4、IL-13、IFN-γ测定.结果 模型组较对照组:IFN-γ值减低[(54.44±14.60)pg/ml vs (85.14±18.17)pg/ml](P＜0.01);IL-4值增高[(26.96±8.95)pg/ml vs (14.37±4.62)pg/ml](P＜0.01);IL-13值增高[(120.52±34.09)pg/ml vs (70.99±22.14)pg/ml](P＜0.01).治疗组较治疗对照组IFN-γ值增高[(95.96±26.86)pg/ml vs (48.53±13.89)pg/ml],(P＜0.01);IL-4值减低[(16.79±7.58)pg/ml vs (30.53±13.34)pg/ml](P＜0.05);IL-13值减低[(95.42±15.17)pg/ml vs (139.25±21.58)pg/ml](P＜0.05).结论 灭活卡介苗治疗对哮喘小鼠有治疗作用,其机制可能与调节Th1/Th2型细胞因子平衡、重建Th1免疫反应及抑制Th2免疫反应有关.     

内毒素在小鼠过敏性哮喘模型所起作用的实验研究

目的建立小鼠过敏性哮喘模型,在过敏原的致敏和激发阶段给予不同剂量内毒素(即脂多糖LPS),探讨内毒素在哮喘模型中的作用机制。方法建立BALB/c小鼠哮喘模型,设立实验组和对照组开展后续实验。实验组包括:鸡卵清白蛋白(OVA)致敏,OVA激发组;OVA致敏,LPS激发组;OVA致敏,OVA+LPS联合激发组;LPS暴露于OVA致敏前,OVA激发组;对照组包括试剂对照组和健康对照组。各实验组的LPS剂量均分为高、中、低三个剂量组。通过测定小鼠气道阻力和肺顺应性来反映其肺功能改变;流式细胞术(FCM)测定FITC、PE阳性细胞数,表征CD4+CD2+5调节性T淋巴细胞(以下简称Treg细胞)的相对比例;荧光实时聚合酶链反应定量测定肺脏和脾脏Foxp3mRNA表达的水平;ELISA法测定血浆总IgE和OVA特异性IgE水平,抗体夹心ELISA法测定支气管肺泡灌洗液(BALF)中Th2细胞因子(IL-4和IL-5)的水平;计数BALF中白细胞总数和嗜酸性粒细胞等各类白细胞所占百分比;常规病理切片观察肺组织改变情况。结果结果表明,与哮喘模型组小鼠相比,在LPS各处理组中,总体上能诱导Treg细胞的产生,改善肺功能,降低IgE滴度,降低Th2介导的免疫反应,同时诱导肺部炎症。尤其是在致敏阶段腹腔注射内毒素可以缓解由过敏原诱导的气道炎症和改善肺功能,诱导肺组织和脾组织Foxp3mRNA表达的增加。结论LPS抑制了Th2介导的过敏反应,可能与Foxp3表达量的增加相关,后者诱导Treg细胞增殖,对哮喘症状起到缓解作用。     

BALB/c小鼠食物过敏动物模型的实验研究

目的　BALB/ c小鼠作为食物过敏动物模型的可行性一直存在争议 ,本研究探讨BALB /c小鼠作为食物过敏动物模型的可行性。方法　对相应试验组动物分别腹腔注射 0 . 2 5ml 2 0mg/ ml卵清蛋白 (OVA)、牛血清白蛋白 (BSA)、大豆胰蛋白酶抑制剂 (TI)和马铃薯酸性磷酸酶 (PAP )共两次 ,间隔为一周。检测血浆中的组胺水平和血清中特异IgE抗体含量 ,同时进行皮肤过敏反应实验 (PCA)。结果　OVA、BSA、TI及PAP组32倍稀释血清中均可检测到特异IgE抗体 ;血浆中组胺水平较对照组显著升高。常见致敏食物蛋白质OVA和TI、不常见致敏食物蛋白质BSA和无致敏史食物蛋白质PAP均可使BALB/ c小鼠产生过敏反应。结论　BALB c小鼠不适合作为食物过敏动物模型。     

Coxsackievirus B3 myocarditis in C3H/HeJ mice: Description of an inbred model and the effect of exercise on virulence

The effect of forced exercise on the development of coxsackievirus B3 myocarditis in inbred C3H/HeJ mice was studied. Four groups of mice (30 per group) were formed: infected-exercised (Group I); infected-unexercised (Group II); uninfected-exercised (Group III); and uninfected-unexercised (Group IV). Infected mice were inoculated intraperitoneally with 1.0 x 10(2.1) TCID50 coxsackievirus B3. Exercised animals were swum daily for 60 minutes on days 1-9. Myocardial viral titers were acutely elevated on day 3 of infection and were augmented significantly by exercise on days 6 and 9. Exercise increased the overall mortality from 0-10% to 20-40%; significantly increased heart: body weight ratios on days 6, 9 and 13; and increased the extent of myocardial fiber necrosis. We have reproduced the acceleration of CB3 myocarditis by exercise in the inbred C3H model.     

Involvement of TLR4 in diazinon-induced neurotoxicity in mice

Our laboratory recently reported that Toll-like receptor (TLR) 4 may play a role in the neurotoxic effects in mice exposed to the environmental toxic chemical toluene. To investigate the role of TLR4 in hippocampal neurotrophin expression, C3H/HeN (TLR4 intact) and C3H/HeJ (TLR4 defective) male adult mice were administered diazinon (0, 0.05, 0.5 or 5 mg/kg) intraperitoneally once a week for three weeks. Twenty-four hours after the final diazinon injection, the hippocampus was collected from each mouse to detect mRNA expression of neurotrophins (nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF)) by the real-time RT-PCR method. There was no difference between groups in neurotrophin expression in the C3H/HeN mice. However, the expression of NGF and BDNF mRNAs was suppressed significantly in the diazinon-injected C3H/HeJ mice compared with their control group. We also found an increased tendency of proinflammatory chemokine CCL3 mRNA and a marked increase in the proapoptotic gene Bax mRNA in the diazinon-injected C3H/HeJ mice. Our findings indicate that diazinon injection affects neurotrophin expression in the hippocampus in TLR4-defective mice but not in TLR4 intact mice. These results suggest that a defective TLR4 signaling pathway in the mouse hippocampus can be easily affected by diazinon administration.     

Adverse cardiovascular effects with acute particulate matter and ozone exposures: interstrain variation in mice

OBJECTIVES: Increased ambient particulate matter (PM) levels are associated with cardiovascular morbidity and mortality, as shown by numerous epidemiology studies. Few studies have investigated the role of copollutants, such as ozone, in this association. Furthermore, the mechanisms by which PM affects cardiac function remain uncertain. We hypothesized that PM and O(3) induce adverse cardiovascular effects in mice and that these effects are strain dependent. STUDY DESIGN: After implanting radiotelemeters to measure heart rate (HR) and HR variability (HRV) parameters, we exposed C57Bl/6J (B6), C3H/HeJ (HeJ), and C3H/HeOuJ (OuJ) inbred mouse strains to three different daily exposures of filtered air (FA), carbon black particles (CB), or O(3) and CB sequentially [O(3)CB; for CB, 536 +/- 24 microg/m(3); for O(3), 584 +/- 35 ppb (mean +/- SE)]. RESULTS: We observed significant changes in HR and HRV in all strains due to O(3)CB exposure, but not due to sequential FA and CB exposure (FACB). The data suggest that primarily acute HR and HRV effects occur during O(3)CB exposure, especially in HeJ and OuJ mice. For example, HeJ and OuJ mice demonstrated dramatic increases in HRV parameters associated with marked brady-cardia during O(3)CB exposure. In contrast, depressed HR responses occurred in B6 mice without detectable changes in HRV parameters. CONCLUSIONS: These findings demonstrate that important interstrain differences exist with respect to PM- and O(3)-induced cardiac effects. This interstrain variation suggests that genetic factors may modulate HR regulation in response to and recuperation from acute copollutant exposures.     

Entamoeba histolytica in inbred mice: evidence of liver involvement

Following observations that certain mouse strains appeared to be susceptible to liver invasion by Entamoeba histolytica, mouse strains CBA/HN, CBA/HN, nu/nu, CBA/HN nu/+, CBA/HN Dh/+, CBA/HN +/+, C3H/HeJ Lps/Lps and AB/H, were infected intra-intestinally with strain SAW 408 (zymodeme II) of the amoeba. A number of mice from each group died during the first 14 days with acute lesions. The remainder were killed at 4 and 6 months after infection. CBA/HN, CBA/HN Dh/+, and CBA/HN +/+ mice all had amoeba-infected livers, although only 2 mice had extensive, typical liver lesions.     

TLR4-dependent adjuvant activity of Neisseria meningitidis lipid A

The adjuvant activity of Neisseria meningitidis serogroup B lipopoly(oligo)saccharide (LOS) from wild-type and genetically defined LOS mutants and unglycosylated meningococcal lipid A was assessed in C3H/HeN and C3H/HeJ mice. Meningococcal lipid A, a weak agonist for TLR4/MD-2 in human macrophages, was found to have adjuvant activity similar to that of wild-type and KDO(2)-lipid A LOS in C3H/HeN mice. All meningococcal LOS structures as adjuvants induced high titers of IgG1, IgG2a and IgG2b but very little IgG3 to OMP compared to no adjuvant PBS controls. In addition, induced OMP antibodies were shown to have high bactericidal activity against serogroup B meningococci. Purified LOS and lipid A structures failed to induce any adjuvant activity in C3H/HeJ mice indicating that meningococcal LOS as an adjuvant was TLR4-dependent. Unglycosylated meningococcal lipid A because of its weak agonist activity for human macrophages and retention of adjuvant activity may be a candidate for use in serogroup B meningococcal OMP and OMV vaccines and for use as an adjuvant in other vaccines.     

Radiation-induced apoptosis in peritoneal resident macrophages of C3H mice

Gamma ray-radiation induced significant apoptosis in peritoneal resident macrophages (PRMs) of C3H/HeJ (C3H) mice, but not in other strains of mice. To investigate the role of DNA damage in the apoptosis, DNA damage was quantified in PRMs by use of the alkaline single-cell gel electrophoresis (Comet) assay. No significant difference was found between C3H and C57Black/6 mice in either radiation-induced DNA damage or repair. Radiation induced apoptosis at the same levels in PRMs of p53 knockout mice and atm knockout mice as those of wild-type C3H mice; however radiation-induced apoptosis was significantly less extensive in the thymocytes of these mutant mice than in those of wild-type mice. Apoptosis was also induced at the same level by an irradiation in PRMs of C3H scid mice as in those of wild-type C3H mice. Therefore it was suggested that radiation-induced DNA damage and TP53, ATM, or DNA-PK-mediated cellular responses occurring downstream thereof were not involved in the radiation-induced apoptotic cell death in C3H mouse PRMs.     

Intestinal amoebiasis: delayed-type hypersensitivity response in mice

Delayed-type hypersensitive (DTH) response was evaluated in C3H/HeJ mice intestinally infected with Entamoeba histolytica. Infected and non-infected control mice were challenged with amoebic antigen on day 5, 10, 15, 20, 25, 30, 40, 50, and 60 post-infection. Maximum footpad swelling was observed after 24 hours of the challenge. The E. histolytica-infected mice exhibited a DTH response on day 5, 15, 20, 25, 40 and 60 post-infection. However, on day 10, 30, and 50, such response was similar to that of the non-infected control mice. The mice developed an evident DTH response late in the course of infection (25 days post-infection). The infected mice did not show any alteration to their DTH response against heterologous unrelated antigen (sheep red blood cells), suggesting that cellular anergy was antigen-specific.     

重组粉尘螨Ⅱ类变应原致敏小鼠肺部变应性炎症模型的建立

目的探讨以重组粉尘螨Ⅱ类变应原(rDerf2)建立小鼠肺部变应性炎症模型的方法。方法将30只6～8周龄雌性SPF级BALB/c小鼠,随机分为PBS组(阴性对照组)、卵清蛋白(OVA)组(阳性对照组)和rDerf2组(实验组)。实验组分别于第0、7、14天用rDerf2对小鼠腹腔进行注射致敏;再于第21天开始,连续7d进行雾化激发;对照组分别用PBS和OVA代替。最后一次雾化激发后24h内处死动物,观察肺组织病理变化、支气管肺泡灌洗液(BLAF)中的白细胞计数及分类,用ELISA测定BLAF和脾细胞培养上清中白细胞介素2(IL-2)、IL-4、IL-17和IFN-γ的含量及血清中IgG1、IgE抗体水平变化。结果实验组和阳性对照组BALB/c小鼠肺组织呈现明显的炎症性病理改变;rDerf2组的BALF中白细胞总数〔(17.39±1.03)×106/L〕及嗜酸性粒细胞(EOS)〔(1.61±0.03)×106/L〕计数,均明显高于PBS组(P<0.01);BALF中IL-4〔(78.92±9.06)pg/ml〕、IL-17〔(201.63±31.26)pg/ml〕与PBS组比较呈明显的高水平表达(P<0.01);而IL-2〔(8.29±1.27)pg/ml〕和IFN-γ〔(51.04±15.85)pg/ml〕的含量则显著下降(P<0.01);上述指标在脾细胞培养上清中出现类似的变化。rDerf2组血清中IgE〔(37.63±6.57)IU/ml〕、IgG1〔(16.68±2.90)μg/ml〕的含量表现为Th2型反应增强趋势;但OVA组和rDerf2组间所有指标差异均无统计学意义(P>0.05)。结论用rDerf2腹腔注射致敏后再雾化激发的方式能够成功构建小鼠肺部变应性炎症模型。