摘要
目的 旨在探讨普通小麦（TA）对卵清蛋白（OVA）致敏小鼠过敏反应的作用及其机制.方法 将25只BALB/c雌鼠随机分为对照组、OVA组、TA 100 mg/kg组、TA 200 mg/kg组和地塞米松（dexamethasone,DEX）组,每组5只.将OVA 20μg与氢氧化铝1 mg溶解于100μl PBS溶液中,并向每只实验组小鼠腹腔内注射以致敏小鼠,而对照组小鼠腹腔内则注射100μl 0.9％生理盐水,饮用水饲养.首次致敏后2周,重复上述操作,并将不同浓度药物稀释于1％CMC溶液中继续饲养,饲养过程中记录每只小鼠的过敏症状与行为评分.首次致敏后12 d对照组、实验组小鼠外耳皮下分别注射20μl 0.9％生理盐水与OVA溶液,6 h、24 h测量每只小鼠外耳厚度,24 h后剪取各组小鼠外耳组织经苏木精-伊红（HE）染色后观察过敏症状.首次致敏后6周,颈椎脱臼法处死小鼠,经腹主动脉采集的血液经离心、取上清后,通过ELISA试剂盒检测IgE、IgG1、TNF-α、IL-4及抗OVA IgE、IgG1了解各炎性细胞因子的分泌情况;取出的脾脏组织提取其淋巴细胞后接种于培养皿,以不同的药物刺激后利用ELISA检测Th1细胞因子（IFN-γ、IL-12）及Th2细胞因子（IL-4、IL-5、IL-13）,利用RT-PCR检测脾淋巴细胞中IFN-γ、IL-4、IL-5、IL-12及IL-13的表达情况.结果 OVA组小鼠的过敏症状行为评分与外耳厚度均显著高于对照组及其他实验组,差异有统计学意义（P〈0.05）,实验组中其与TA呈剂量依赖性降低,组织病理学结果也证实了上述结果.ELISA结果显示,实验组小鼠血浆中IgE、IgG1、IL-4及TNF-α水平与对照组比较显著升高,其水平与TA呈剂量依赖性降低,尤其是IgE、TNF-α在TA高浓度组显示出与DEX组相似的抑制效果.RT-PCR结果显示,实验组较对照组Th1细胞因子（IFN-γ、IL-12）的含量显著增加,差异有统计学意义（P〈0.05）,其水平与TA未显示出剂量依赖性效应,而DEX组显示出明显的抑制作用.实验组Th2细胞因子（IL-4、IL-5及IL-13）的表达显著高于对照组,差异有统计学意义（P〈0.05）,TA组的IL-4、IL-5、IL-13表达均较OVA组有不同程度的降低,尤其在TA 100μg/ml组中其表达较OVA组分别降低了约60％、18％与20％,显示出了明显的抑制作用.结论 TA可能通过选择性抑制Th2细胞因子（IL-4、IL-5、IL-13）及IgE、IgG,而未抑制Th1细胞因子（IFN-γ、IL-12）,在保持体内免疫平衡的基础上有效降低过敏相关免疫应答,从而发挥抗过敏作用.本研究为新型抗过敏治疗药物（TA）的开发提供理论基础和实验依据.
Objective To investigate the inhibitory effects and mechanism of Triticum aestivum （TA） on anaphylaxis in ovalbumin （OVA）-sensitized mice. Methods Twenty-five female BALB/c mice were randomly divided into five groups （n = 5 ）, including control group, OVA group, 100 mg/kg TA group, 200 mg/kg TA group and DEX （dexamethasone） group. OVA （20μg） and aluminum hydroxide （1 rag） were dissolved in 100 μl of PBS solution and injected into the abdominal cavity of each experimental mouse, while the mice in the control group were injected with 100μl of 0.9% normal saline. All mice were given drinking water. The above processes were repeated two weeks later, and different concentrations of experimental drugs were diluted in 1% CMC （carboxymethyl cellulose） solution to feed mice. Allergic symptoms and behaviors of each mouse were scored in the process of feeding. On the 12th day after first sensitization, mice in the control and experimental groups were subcutaneously injected in the auricle with 20μl of 0.9% saline solution and OVA, respectively. The thickness of each mouse＇s auricle was measured 6 and 24 hours later. Histopathologic changes in auricle tissues were observed by hematoxylin-eosin（HE） staining 24 hours later. The mice were euthanized by cervical dislocation in the 6th week after first sensitization. Abdominal aorta serum samples were collected and tested for specific inflammatory cytokines （IgE, IgGl, TNF-α, IL- 4） and anti-OVA antibodies （IgE, IgG1 ） by ELISA. ELISA and RT-PCR were used to detect the expression of IFN-γ, IL4, IL-5, IL-12 and IL-13 after spleen lymphocytes were stimulated with different concentrations of drugs. Results Both the score of allergic symptoms and behavior and the thickness of the auricle of the OVA group were the highest among all groups （ P〈0. 05 ）. A TA dose-dependent decrease was found in both of the two parameters, which was confirmed by histopathnlogical analysis. ELISA results showed that the plasma levels of IgE, IgG1, IL-4 and TNF-α in the experimental groups were significantly higher than those in the control group, but all of them were decreased in a TA dose-dependent manner. TA at the high concentration of 200 mg/kg was similar to DEX in inhibiting the expression of IgE and TNF-α. RT-PCR resuhs showed that Thl cytokines （IFN-γ and IL-12） in the experimental groups increased significantly as compared with those in the control group （P〈0.05）. Expression of IFN-γ and IL-12 was not significantly affected by TA at various concentrations, but markedly inhibited by DEX. The levels of Th2 cytokines （IL-d, IL-5 and IL-13） in the experiment groups were significantly higher than those in the control group （P〈0.05）. Compared with the OVA group, the levels of IL-4, IL-5 and IL-13 were significantly reduced in TA groups, es- pecially in the TA 100μg/ml group in which the expression of the three cytokines was decreased by about 60%, 18% and 20%, respectively. Conclusion TA helps to maintain immune balance by selectively inhibiting the expression of Th2 cytokines （ IL-4, IL-5, IL-13）, IgE and IgG and not influencing Thl cyto- kines （IFN-3, and IL-12）. It has an anti-allergic effect as it effectively suppresses allergic responses. This research provides both theoretical and experimental basis for further development of novel anti-anaphylactic treatment with TA.
作者
金永龙
刘希光
肖文静
宋浩
崔银实
朴春姬
Jin Yonglong;Liu Xiguang;Xiao Wenfing;Song Hao;Cui Yinshi;Piao Chunji(Department of Radiation Oncology,the Affiliated Hospital of Qingdao University,Qingdao 266000,China)
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
2018年第8期572-581,共10页
Chinese Journal of Microbiology and Immunology
基金
山东省医药卫生科技发展计划项目（2017WS126）
