细菌溶解产物对支气管哮喘小鼠TGF-β1及Foxp3表达的影响

Impact of bacterial lysates on the expression of TGF-β1 and Foxp3 in mouse

目的 建立支气管哮喘（简称哮喘）小鼠模型,给予细菌溶解产物（OM-85BV）干预,通过动物实验探讨免疫调节剂-OM-85BV干预对哮喘小鼠外周血及外周免疫器官-脾脏Treg功能相关细胞因子TGF-β1及特异性转录因子Foxp3的表达的影响.方法 清洁级4～6周龄BALB/c小鼠48只,随机分为6组.a组：空白对照组;b组：正常OM-85BV对照组;c组：哮喘模型组;d组：地塞米松（Dex）干预组;e组：OM-85BV干预A组;f组：OM-85BV干预B组（干预时间较e组延长10 d）.在实验第1天、第8天和第15天分别给c、d、e、f4组小鼠腹腔注射卵清白蛋白（OVA）-Al（OH）3悬液使其致敏,其余组以PBS缓冲液对照.第17天至第26天给f组小鼠OM-85BV-生理盐水（NS）溶液灌胃,其余5组以NS模拟灌胃.第27天至第31天分别给c、d、e和f组OVA-NS溶液滴鼻激发,其余两组以等量NS对照.其中b、e和f组在激发前1h给予OM-85BV干预,d组给予Dex干预,其余组用NS对照.末次激发后24 h麻醉并解剖小鼠,心脏采血测定血清TGF-β1水平,取部分右肺行HE染色作病理学检查,取胸腺组织行免疫组织化学的方法测定其中Foxp3的表达,取脾脏行Real time-PCR测调节性T细胞（Treg）相关细胞因子TGF-β1 mRNA及Foxp3 mRNA的表达.结果 与a组对照相比较,c组小鼠支气管痉挛收缩,支气管上皮增生紊乱,支气管周围可见以淋巴细胞和嗜酸性细胞为主的炎症细胞浸润,管腔内有少量黏液和脱落的上皮细胞,而且肺泡间隔增宽;c组小鼠血清中TGF-β1水平较a组显著降低,d、f组较c组情况明显增加,e组次之,差异有统计学意义（P＜0.01）.胸腺组织免疫组织化学结果显示各组之间Foxp3的表达差异无统计学意义（P＞0.05）,但是实验过程中可以观察到,d组胸腺体积较其他各组明显缩小.c组脾脏TGF-β1 mRNA的相对表达显著低于正常组,f组较c组表达明显增加,d组次之,差异有统计学意义（P＜0.01）.c组脾脏Foxp3 mRNA的相对表达显著低于正常组,f组较c组表达明显增加,e组、d组次之,差异有统计学意义（P＜0.01）.结论 口服OM-85BV干预有利于改善哮喘小鼠模型气道炎症,显著提高外周血及外周免疫器官-脾脏的Treg功能相关细胞因子TGF-β1及特异性转录因子Foxp3的表达,二者可能是预防哮喘气道炎症的发生和发展的治疗靶点.

Objective To establish mouse allergic bronchial asthma （asthma） model and observe the effect of bacterial lysates （OM-85BV） on airway inflammation,as well as the expression of TGF-β1 and Foxp3 in serum and spleen.Methods Forty-eight 4 to 6 weeks healthy male BALB/c mice were used as research subjects and randomly divided into six groups,a：contral group,b：OM-85BV contral group,c：allergic asthma model,d：dexamethasone group （Dex group）,e：OM-85BV A group,f：OM-85BV B group （the intervention time was prolonged 10 days than group e）.BALB/c mice were sensitized and challenged by ovalbumin （OVA）.Mice in groups c,d,e and f were intraperitoneally administered by antigen （OVA）-Al（OH）3 on days 1,8 and 15,others were administered by PBS.From the 17th day to the 26th day,mice in the group f were treated with OM-85BV and others were treated with normal saline.In the next days,mice in groups c,d,e and f were intranasal given OVA for 5 consecutive days.Additionally,mice in groups b,e and f were treated with OM-85BV before challenge,while mice in the group d were administered by Dex,others were treated with normal saline at the same dose.Twenty four hours after the last intranasal administration,mice were anesthetized and dissected.The TGF-β1 levels of blood serum were detected.The removed parts of lung tissue were for histological to observe airway inflammation and the thymuses were prepared for immunohistochemistry examination to detect the expression of Foxp3.The expression of TGF-β1 mRNA and Foxp3 mRNA in spleen were detected by Real time-PCR.Results Compared with groups a and b,lung tissue biopsies by HE staining from the asthma group showed obvious airway inflammation.The situation of groups d and f was significantly improved than group c while the differences between groups e and c were not evidently.TGF-β1 levels from the asthma group in blood serum were significantly lower than groups a and b while the levels of groups d and f were significantly improved than group c.The immunohistochemistry examination of Foxp3 in thymus didn＇t show significant difference,but thymuses of group d were obviously smaller than other groups.Compared with the contral group （1.001＆±0.000）,the levels of TGF-β1 mRNA from the asthma group （0.851±0.071） were significantly lower.The corresponding levels of groups d （1.085 ± 0.042） and f （1.292 ± 0.083） were significantly improved than group c （P 〈0.01）.Compared with the contral group （1.001 ±0.000）,the levels of Foxp3 mRNA from the asthma group （0.585 ± 0.192） were significantly lower.The corresponding levels of group f （2.074±0.037） were significantly improved than group c （P 〈0.01）.Conclusions Administered by OM-85BV helps reduce airway inflammation in asthmatic mice and significantly improve the expression of TGF-β1 and Foxp3 of the peripheral blood and peripheral immune organs.The two may be the therapeutic targets to prevent the occurrence and development of airway inflammation in asthma.