共刺激分子B7H3在支气管哮喘中的作用研究

硕士学位论文 论文题目 共刺激分子 B7-H3 在支气管哮喘中的 作用研究 研究生姓名 陈 赢 指导教师姓名 季 伟 （ 教授 ） 专 业 名 称 儿科学 研 究 方 向 呼 吸 论文提交日期 2013年 4月 共刺激分子 B7-H3在支气管哮喘中的作用研究 中文摘要 I 共刺激分子 B7-H3 在 支气管哮喘 中 的作用研究 中文摘要 第一部分 可溶性 B7-H3 在支气管哮喘 患儿血浆 中的 表达及临床意义 目的： 研究支气管哮喘患 儿 外周血中 可溶性 B7-H3（ sB7-H3） 的表达水平，并分析其与细胞因子 水平 的相关性，探讨 sB7-H3 的异常表达在支气管哮 喘 急性发作 中的临床意义。 方法： 选取 2012 年 1 月 2012 年 12 月在苏州大学附属儿童医院呼吸科住院的支气管哮喘患儿 21 例，并选取 外科择期手术 （ 腹股沟斜疝、血管瘤 、腺样体肥大 ）而住院的 患儿 16 例作为对照组 儿童 ，分为支气管哮喘急性发作期组 （ A 组 ） 、恢复期组 （ B组） 及对照组 （ C组） 。同时采集支气管哮喘患儿急性发作期及恢复期的外周血，以及 外科择期手术 患儿的外周血， 采用酶联免疫吸附试验（ ELISA） 检测血浆中 sB7-H3、 IFN-、 IL-4 及 IL-10的表达水平，及 分析 支气管哮喘患儿 急性发作期 血浆 中 sB7-H3的 水平与细胞因子 IFN-、 IL-4、 IL-10的相关性。 结果： 哮喘急性发作期 组血浆中 sB7-H3 的水平明显高于 恢复期 组及 对照 组（ P0.05） ，而 恢复期 组虽高于 对照 组，但无统计学差异； 哮喘急性发作期 组血浆 中IFN-、 IL-10水平 均 低于恢复期 组及对照组 （ P0.05）， 恢复期与对照组相比无统计学差异 ；血浆 中 IL-4的水平 在 哮喘急性发作期 组与 恢复期 组均高于 对照 组（ P0.05），且 哮喘急性发作期 组的表达水平高于 恢复期 组（ P0.05）。支气管哮喘 患儿 急性发作期 血浆 中 sB7-H3的表达 水平 与 IL-4的水平具 有正 相关 性，与 IFN-、 IL-10的 水平 无相关性。 结论： sB7-H3在支气管哮喘急性发作期异常 高 表达 ； sB7-H3可能通过增强 Th2细胞因子的分泌而在支气管哮喘 的 急性发作期起作用。 关键词： sB7-H3；支气管哮喘； Th2细胞；细胞因子 中文摘要 共刺激分子 B7-H3在支气管哮喘中的作用研究 II 第二部分 抗 B7-H3 单克隆抗体对哮喘小鼠模型的作用研究 目的 ： 探讨抗 B7-H3单克隆抗体对哮喘小鼠模型诱导期及效应期的治疗作用。 方法 ： 6-8周龄雌性 BALB/c小鼠（ 18-20g），随机均分为 5组， 每组 10只， 分别为哮喘组（ A组），生理盐水对照组（ B组）， IgG组（ C组），抗 B7-H3抗体诱导期 组（ D组），抗 B7-H3抗体效应期 组 （ E组 ） 。 A、 C、 D、 E组均给予 OVA致敏及激发（ 0、 10天给予 OVA腹腔注射致敏， 第 20天给予 OVA雾化吸入，每天半小时连续 7 天）， C 组、 D 组在第 2， 7， 11， 14， 18 天分别给予腹腔注射 IgG、抗B7-H3抗体， E组在第 2， 7， 11， 14， 18天给予腹腔注射 IgG， 第 21， 24， 26天腹腔注射抗 B7-H3抗体（ OVA雾化 1小时前注射）。 B组给予同剂量生理盐水 注射、激发。各组小鼠于末次激发 2小时后 麻醉 处死，采集标本。小鼠进行肺泡灌 洗，肺泡灌洗液（ BALF）进行细胞 总数 计数及分类计数；并采用 ELISA测定小鼠 BALF中的IFN-、 IL-4、 IL-17的水平；小鼠肺组织切片行 HE染色、 PAS染色及免疫组化法（ SABC法），观察肺组织炎 性 细胞及嗜酸性细胞浸润情况、 气道 粘液分泌情况及 B7-H3 表达情况。 结果 ： 哮喘组肺泡灌洗液（ BALF）中的细胞总数及嗜酸性细胞数 多于生理盐水对照组 及 抗 B7-H3抗体诱导期组 ，差异具有统计学意义（ P0.05） ， 哮喘组、 IgG组、抗 B7-H3 抗体效应期组 BALF 中 细胞总数及嗜酸性细胞数 比较差异无统计学意义，抗 B7-H3 抗体诱导期组 BALF 中的细胞总数及嗜酸性细胞数高于生理盐水对照组，差异具有统计学意义（ P0.05）。哮喘组、 IgG组、抗 B7-H3抗体诱导期组及抗 B7-H3抗体效应期组 BALF中的 IFN-的水平均低于生理盐水对照组（ P0.05），哮喘组、IgG 组、抗 B7-H3 抗体 效应 期组 IFN-的水平 无统计学差异， 抗 B7-H3 抗体诱导期组 IFN-的水平 高于 IgG组（ P0.05） 。 哮喘组、 IgG组、 抗 B7-H3抗体诱导期组 及抗 B7-H3 抗体效应期组 BALF 中的 IL-4、 IL-17 的水平均高于生理盐水对 照组（ P0.05），哮喘组、 IgG 组、抗 B7-H3 抗体 效应 期组 IL-4、 IL-17 水平无统计学差异 ， 抗 B7-H3抗体 诱导 期组 IL-4、 IL-17水平低于 IgG组（ P0.05）。肺组织病理染色显示哮喘组、 IgG组、抗 B7-H3抗体效应期组炎性细胞、嗜酸性细胞浸润及粘液分泌较抗 B7-H3 抗体诱导期组明显，生理盐水对照组无明显炎性细胞、嗜酸性细胞浸共刺激分子 B7-H3在支气管哮喘中的作用研究 中文摘要 III 润、粘液分泌。免疫组化显示哮喘组、 IgG组 B7-H3高表达，生理盐水对照组 B7-H3不表达，抗 B7-H3抗体诱导期组、抗 B7-H3抗体效应期组 B7-H3部分表达。 结论 ： 哮喘的诱导期给予抗 B7-H3 单克隆抗体治疗能通过抑制 Th2 型细胞因子（ IL-4） 和 Th17 型细胞因子（ IL-17）的分泌，同时促进 Th1 型细胞因子（ IFN-）的分泌，从而减轻 OVA致敏哮喘小鼠的气道病理改变。 关键词 :抗 B7-H3单克隆抗体；哮喘小鼠模型；细胞因子 作 者 ：陈 赢 指导老师 ：季 伟 Abstract Role of costimulatory molecule B7-H3 in bronchial asthma IV Role of costimulatory molecule B7-H3 in bronchial asthma Abstract Expression and clinical significance of the soluble B7-H3 in plasma of children with bronchial asthma Objective: To discuss the level of soluble B7-H3 in the peripheral blood of children with bronchial asthma, and analyze its correlation with the expression of cytokines, to explore the clinical significance of abnormal expression of soluble B7-H3 in acute phase of bronchial asthma. Methods: 21 cases of children with bronchial asthma were enrolled at department of respiration of Childrens hospital affiliated in Soochow university from January 2012 to December 2012, and 16 cases of children (Indirect inguinal hernia, hemangioma, adenoid hypertrophy) hospitalized in surgery as control group children, divided into acute phase of asthma group (group A), recovery phase group (group B) and control group (group C). At the same time, peripheral blood were collected in children with bronchial asthma in acute phase and recovery phase as well as children in surgery, Enzyme-linked immunosorbent assay were used to detect the level of soluble B7-H3, IFN-gamma, interleukin-4 and interleukin-10 in plasma, and analyze the correlation between the level of soluble B7-H3 and cytokines IFN-gamma, interleukin-4, interleukin-10 in plasma in acute phase of asthma. Results: The level of soluble B7-H3 in plasma in acute phase of asthma group were significantly higher than the recovery phase group and control group (P0.05), while recovery phase group was higher than control group, but no significant difference, the level of IFN-gamma and interleukin-10 in acute phase of asthma group were lower than that of recovery phase group and control group (P0.05), recovery phase group and control group had no significant difference. The level of interleukin-4 in plasma both in acute phase of asthma group and recovery phase group were higher than that of control group (P0.05), and the level in acute phase of asthma group was higher than that of recovery phase group Role of costimulatory molecule B7-H3 in bronchial asthma Abstract V (P0.05). The level of soluble B7-H3 and the level of interleukin-4 in plasma has positive correlation in children in acute phase of bronchial asthma, and has no correlation with IFN-gamma, interleukin-10. Conculsion: The level of soluble B7-H3 was abnormal high expression in acute phase of bronchial asthma, soluble B7-H3 may enhance the secretion of Th2 type cytokines and has effect in acute phase of bronchial asthma. Keywords: soluble B7-H3； bronchial asthma； Th2 cell； cytokine Role of anti B7-H3 monoclonal antibody in mouse model of asthma Objective: To explore the therapeutic function of anti B7-H3 monoclonal antibody in mouse model of asthma in induction phase and effector phase. Methods: Female BALB/c mice (weight 18-20g and 6-8 wk old) were divided into five groups, every group has 10 mice, asthmatic group (group A), normal saline control group (group B), IgG group (group C), anti B7-H3 antibody induction group (group D), anti B7-H3 antibody effector group (group E). Mice in group A, group C, group D and group E were given OVA sensitization and excitation (mice were sensitized to OVA by intraperitoneal injection on days 0 and 10, then they were exposed to OVA aerosol challenges for thirty minutes on consecutive seven days on day 20), group C and group D were given intraperitoneal injection of IgG and anti B7-H3 antibody on days 2, 7, 11, 14, 18, group E were given intraperitoneal injection of IgG on days 2, 7, 11, 14, 18, intraperitoneal injection of anti B7-H3 antibody on days 21, 24, 26 (intraperitoneal injection before one hour of OVA challenges). Mice in group B were given saline as above methods. After two hour of last challenge all mice were anesthetized to death to collect samples, all mice were alveolared lavage, and bronchoalveolar lavage fluid (BALF) were collected to count cells number and classify cells, measure the level of IFN-gamma, interleukin-4, interleukin-17 in BALF by Enzyme-linked immunosorbent assay, mouse lung tissue sections stained with HE, PAS and immunohistochemical method (SABC method), to observe the infiltration of inflammatory cells and eosinophilic cells, mucus secretion and the expression of B7-H3 in lung tissue. Results: The total cells and eosinophilic cells in bronchoalveolar lavage fluid in asthmatic group were more than the normal saline control group and anti B7-H3 antibody Abstract Role of costimulatory molecule B7-H3 in bronchial asthma VI induction group, the difference were statistically significant (P0.05). The total cells and eosinophilic cells in bronchoalveolar lavage fluid in asthmatic group, IgG group and anti B7-H3 antibody effector group were no statistically significant. The total cells and eosinophilic cells in anti B7-H3 antibody induction treatment group were more than the normal saline control group (P0.05). The level of IFN-gamma in BALF in asthmatic group, IgG group, anti B7-H3 antibody induction group and anti B7-H3 antibody effector group were lower than the normal saline control group (P0.05), the level of IFN-gamma in asthmatic group, IgG group and anti B7-H3 antibody effector group were no statistically significant, the level of IFN-gamma in anti B7-H3 antibody induction group were higher than IgG group (P0.05). The level of interleukin-4 and interleukin-17 in asthmatic group, IgG group, anti B7-H3 antibody induction group and anti B7-H3 antibody effector group were higher than the normal saline control (P0.05), the level of interleukin-4, interleukin-17 in asthmatic group, IgG group and anti B7-H3 antibody effector group were no statistically significant, the level of interleukin-4, interleukin-17 in anti B7-H3 antibody induction group were lower than IgG group (P0.05). Lung tissue pathological stained showed more infiltration of inflammatory cells, eosinophilic cell and mucus secretion in asthmatic group, IgG group, anti B7-H3 antibody effector group than that of anti B7-H3 antibody induction group, normal saline control group no significant infiltration of inflammatory cells, eosinophilic cell and mucus secretion. Immunohistochemistry showed high expression of B7-H3 in asthmatic group and IgG group, normal saline control group did not express B7-H3, and B7-H3 partly expression in anti B7-H3 antibody induction group and anti B7-H3 antibody effector group. Conculsion: Treatment with anti B7-H3 monoclonal antibody in induction phase of asthma can inhibit secretion of Th2 type cytokine (interleukin-4) and Th17 type cytokines (interleukin-17), and promote secretion of Th1 type cytokine (IFN-gamma), there by reducing the airway pathological change of mouse model of asthma induced by OVA. Keywords: Anti B7-H3 monoclonal antibody； mouse model of asthma； cytokine Written by: Chen Ying Supervised by: Ji Wei 目 录 前 言 . 1 第一部分 可溶性 B7-H3（ sB7-H3）在支气管哮喘患儿血浆中的表达及临床意义 . 6 材料与方法 . 7 结 果 . 10 讨 论 . 12 参考文献 . 15 第二部分 抗 B7-H3单克隆抗体对哮喘小鼠模型的作用研究 . 18 材料与方法 . 18 结 果 . 23 讨 论 . 29 参考文献 . 33 结 论 . 35 综 述 共刺激分子 B7-H3的生物学作 用及与支气管哮喘的研究进展 . 36 缩略词表 . 45 攻读硕士期间公开发表的论文 . 47 致 谢 . 48 共刺激分子 B7-H3在支气管哮喘中的作用研 究 前 言 1 前 言