摘要
目的应用卵白蛋白(OVA)建立特异性免疫治疗小鼠模型,探讨特异性免疫治疗对支气管哮喘(简称哮喘)小鼠树突细胞(DC)表面分子CD80、CD86表达的影响。方法120只BALB/c小鼠按随机数字表法分为哮喘模型组(A组)40只:用0.1%OVA 10μg连续腹腔注射(共70μg)及1%OVA雾化吸入(共300 mg);免疫治疗模型组(B组)40只:用与A组同剂量的OVA致敏和激发,同时连续尾根部皮下注射OVA 1 mg;对照组(C组)40只:以磷酸盐缓冲液代替OVA,其余同A组。留取各组肺组织切片,经苏木精-伊红(HE)染色观察炎症反应;用酶联免疫吸附测定(ELISA)法检测血清OVA特异性免疫球蛋白IgE(sIgE)及脾脏T淋巴细胞中白细胞介素2(IL-2)和IL-4的分泌。分离各组小鼠脾脏DC,用流式细胞仪检测其表面CD80、CD86分子表达。分离正常小鼠脾脏T淋巴细胞,与上述各组DC共培养;用ELISA法检测T淋巴细胞IL-4、IL-5的分泌量;用3H-胸腺嘧啶核苷(3H-TdR)掺入法检测其增殖反应。结果(1)B组小鼠肺组织中支气管及血管周围以大量淋巴细胞和嗜酸粒细胞为主的炎性细胞浸润明显轻于A组,但并未完全消失;A组血清sIgE吸光度(A)值为712±129,B组为124±59,C组为20±13,A、C组间比较差异有统计学意义(P<0.05),B、C组比较差异无统计学意义(P>0.05)。B组T淋巴细胞分泌IL-2、IL-4水平分别为(8±3)、(8.4±4.3)pg/m l,A组分别为(22±8)、(32.4±12.1)pg/m l,C组分别为(6±4)、(5.1±1.1)pg/m l,A、B两组比较差异有统计学意义(P<0.05),B、C组间比较差异无统计学意义(P>0.05);(2)B组DC表面CD86、CD80阳性表达率分别为58.23%、95.63%,A组分别为77.59%、96.98%,C组分别为77.37%、77.84%;(3)与B组DC共培养的正常小鼠T淋巴细胞体外经OVA刺激后,IL-4、IL-5水平分别为(10.8±2.3)、(18.8±3.8)pg/m l,A组分别为(17.3±4.7)、(35.7±7.9)pg/m l,C组分别为(5.7±2.7)、(11.0±2.2)pg/m l,A、B两组比较差异有统计学意义(P<0.05),B、C组间比较差异无统计学意义(P>0.05);B组DC与正常小鼠T淋巴细胞共培养时,刺激指数(SI)为3.8±0.7,A组为11.5±3.2,C组为5.8±1.5,A、B组间比较差异有统计学意义(P<0.05);B、C组间比较差异无统计学意义(P>0.05)。结论建立了OVA特异性免疫治疗小鼠模型;DC表面CD86分子表达的下调可能是OVA特异性免疫治疗诱导T淋巴细胞功能丧失的机制之一。
Objective To establish a mouse model of ovalbum in （OVA） specific immunotherapy, and to explore the effects of specific immunotherapy on the expression of CD80 and CD86 on spleen-derived dendritic ceils （DC） in a mouse asthmatic model Methods A hundred and twenty BALB/c mice were randomly divided into three groups, with 40 mice each. The asthma model （group A） was established by intraper itoneal, injection of 10 μg OVA and 1% OVA aerosol challenges. Consecutive subcutaneous injection of 1 mg OVA in the dorsal aspect of the foot was then given to establish the model of OVA specific immunotherapy （group B）, while PBS （0.01 mol/L） was given in group C to serve as the control. The pathological changes of lung tissues were studied by HE stain. The serum OVA-specific IgE level, and IL-2 and IL-4 production in the splenic T cells were determined by enzyme-linked immunosorbent assay （ELISA）. The expression of CD80 and CD86 on DC, which were isolated from the spleen, were detected by fluorescein-actived cell sorter. ^3H-thymidine （^3H-TdR） incorperation assay was used to determine the response of splenic T and the secretion of interleukin-4 （IL-4） and IL-5 was assayed with the use of ELISA after coculture of T cells with DC. Results （ 1 ） The infiltration of eosinophils and lymphocytes within epithelium and lamina propria was present in group B, but was milder than that in group A. The absorbance at 490 nm （A490 ） of serum specific IgE in group A 712 ±129, was more than that in group C （20 ± 13, P 〈 0.05）, but there was no significant difference between group B （ 124± 59, P 〉 0.05） and C. The production of IL-2 by splenic T cells in group B （ 8 ±3 ） pg/ml was less than that in group A [ （ 22± 8 ） pg/ml,P 〈 0. 05 ], but there was no significant difference between group B and group C [ （6± 4） pg/ml, P 〉 0. 05 ]. The production of IL-4 by splenic T cells in group B （ 8.4 ±4. 3 ） pg/ml was less than that of group A [ （32. 4 ± 12. 1 ） pg/ml, P 〈 0. 05 ], but there was no significant difference between group B and group C [ （5.1 ± 1.1 ） pg/ml, P 〉 0. 05 ]. （2） The expression of CD86 on splenic DC cells from group B （58.23%） was significantly lower than that from group A （77.59%） and group C （77.84%）, while the expressions of CD80 in group A （96. 98% ） and group B （95.63%） were higher than that in group C （77.37%）. （3） When untreated T cells were cocultured with DC from the individual groups and stimulated by OVA, the secretion of IL-4 from T cells in group B [ （ 10. 8±2. 3） pg/ml] was significantly lower than that in group A [ （ 17.3 ± 4. 7 ） pg/ml, P 〈 0.05 ], but there was no significant difference when compared to group C [ （5.7 ±2. 7 ） pg/ml, P 〉 0. 05 ], and the secretion of IL-5 from T cells in group B [ （ 18. 8 ± 3.8 ） pg/ml] was significantly lower than that in group A [ （ 35.7± 7.9 ） pg/ml, P 〈 0.05 ], but there was no significant difference when compared to group C [ （ 11.0 ± 2. 2） pg/ml, P 〉 0.05 ]. When untreated T cells were cocultured with DC from the individual groups and stimulated by OVA, the stimulating index of T cells in group B （ 3. 8 ± 0. 7 ） was significantly lower than that in group A （ 11.5 ±3.2, P 〈 0. 05 ）, but there was no significant difference when compared to group C （5.8± 1.5, P 〉 0. 05 ）. Conclusions A mouse model of asthma-specific immunotherapy was successfully established. Downregulation of CD86 on the surface of DC might be the underlying mechanisms by which specific immunotherapy promotes Th1 to Th2 polarization and induces T cell anergy.
出处
《中华结核和呼吸杂志》
CAS
CSCD
北大核心
2006年第3期171-175,共5页
Chinese Journal of Tuberculosis and Respiratory Diseases
基金
国家自然科学基金资助项目(30170403)
