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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 specific immunotherapy with ovalbumin (OVA) and investigate the effect of specific immunotherapy on the expression of CD80 and CD86 on dendritic cells in asthmatic mice. Methods A total of 120 BALB / c mice were randomly divided into asthma model group (group A) 40: 0.1% OVA 10μg intraperitoneal injection (70μg total) and 1% OVA inhalation (total 300 mg); 40 mice in the immunotherapy model group (group B) were sensitized and challenged with OVA at the same dose as group A, and 1 mg of OVA was injected subcutaneously at the base of the tail continuously. 40 rats in the control group (group C): OVA was replaced by phosphate buffered saline The rest with A group. The lung tissue sections of each group were collected and the inflammation reaction was observed by hematoxylin-eosin (HE) staining. Serum OVA-specific IgE (sIgE) and splenic T lymphocytes were detected by enzyme linked immunosorbent assay (ELISA) Interleukin 2 (IL-2) and IL-4 secretion. The spleen DC of each group was isolated and the expression of CD80 and CD86 on the surface was detected by flow cytometry. The splenic T lymphocytes of normal mice were isolated and co-cultured with DC of the above groups. The secretion of IL-4 and IL-5 of T lymphocytes was detected by ELISA. 3H-thymidine incorporation Detection of proliferative response. Results (1) Inflammatory cells infiltrating bronchus and perivascular area in bronchus and perivascular area in group B were lighter than those in group A, but did not disappear completely. Serum sIgE absorbance (A ) Was 712 ± 129, in group B was 124 ± 59, in group C was 20 ± 13, there was significant difference between group A and C (P <0.05), but there was no significant difference between group B and C (P > 0.05). The levels of IL-2 and IL-4 secreted by T lymphocytes in group B were (8 ± 3) and (8.4 ± 4.3) pg / ml, and those in group A were (22 ± 8) and (32.4 ± 12.1) pg / (P <0.05). There was no significant difference between B and C groups (P> 0.05). There was no significant difference between B and C groups (P> 0.05). (2) The positive expression rates of CD86 and CD80 on DCs in group B were 58.23% and 95.63% respectively, 77.59% and 96.98% in group A and 77.34% and 77.84% in group C respectively The levels of IL-4 and IL-5 in normal mice co-cultured with OVA stimulated by OVA were (10.8 ± 2.3) and (18.8 ± 3.8) pg / ml, respectively, and those in group A were (17.3 ± 4.7) , (35.7 ± 7.9) pg / ml in group C and (5.7 ± 2.7) and (11.0 ± 2.2) pg / ml in group C respectively. There was significant difference between group A and group B There was no significant difference between the two groups (P> 0.05). The co-culture of DCs in group B with normal mice showed that the stimulation index (SI) was 3.8 ± 0.7, the group A was 11.5 ± 3.2 and the group C was 5.8 ± 1.5 There was significant difference between A and B groups (P <0.05). There was no significant difference between B and C groups (P> 0.05). Conclusions A mouse model of OVA-specific immunotherapy was established. Down-regulation of CD86 expression on DC surface may be one of the mechanisms of OVA-specific immunotherapy-induced loss of T lymphocyte function.