基质细胞衍生因子在哮喘小鼠气道中的表达
作者：黄芸，欧阳海峰，彭晓丹，王文雅，吴昌归
【摘要】 目的观察基质细胞衍生因子-1(stromal-derived factor-1, SDF-1)在鸡卵清蛋白(OVA)诱导小鼠哮喘模型中的表达，探索哮喘气道重塑的发生机制。方法40只雌性SPF级C57BL/6小鼠，体重18～22g，随机分为哮喘组和对照组。按照文献方法建立OVA小鼠哮喘模型。两组动物于末次雾化结束后24h处死，取肺组织行病理切片，苏木精-伊红染色(HE)观察肺脏的组织病理学变化;逆转录聚合酶链式反应(RT-PCR)、免疫印迹法(Western blot)、免疫组织化学(immunohistochemistry, IH)技术检测肺组织SDF-1的表达。结果①哮喘组支气管上皮细胞脱落、气道壁增厚，细支气管和血管周围有大量炎细胞浸润;②哮喘组肺组织SDF-1在RT-PCR、Western blot及IH中均为阳性表达，而对照组则否。结论小鼠哮喘时肺组织分泌大量细胞趋化因子SDF-1，可能是哮喘气道重塑的重要因素之一。
【关键词】 基质细胞衍生因子;哮喘;气道重塑
ABSTRACT: ObjectiveTo study the expression of stromal-derived factor-1 (SDF-1) in the airway of mice with allergic asthma induced by ovalbumin (OVA) so as to explore the mechanism of asthmatic airway remodeling.MethodsForty female C57BL/6 mice of SPF grade were randomly divided into asthma group and control group with 20 in each. Mice were sensitized and challenged with ovalbumin to establish the asthmatic model. All the mice were killed 24 hours after the last aerosolisation. The paraffin embedded sections of lung tissues were stained with hematoxylin and eosin (HE) for analyzing pathological changes. SDF-1 mRNA extracted from lung tissues was assessed by RT-PCR while SDF-1 protein extracted from lung tissues was assessed by Western blot and immunohistochemistry (IH).Results① After repeated allergen challenge, obvious infiltration of inflammatory cells and proliferation of goblet cells and smooth muscles appeared in mouse bronchi. ② According to RT-PCR and Western blot and IH results, the expression of SDF-1 was positive in asthma group but negative in control group.ConclusionA large amount of SDF-1 is secreted in the lung tissues in asthmatic mice, which may be one of the critical factors in asthmatic airway remodeling.
KEY WORDS: stromal-derived factor-1; asthma; airway remodeling
支气管哮喘是一种慢性气道炎症，伴有气道高反应性和气道重塑，而气道重塑则是一个在慢性炎症基础上的异常损伤修复过程。基质细胞衍生因子-1(stromal-derived factor-1, SDF-1)是由基质细胞产生的一种CXC型趋化因子。近年来研究表明，基质细胞衍生因子对组织或器官的创伤、炎症损伤修复起到了重要的作用。但是，SDF-1与哮喘相关性的研究目前还未见报道，我们就此进行了探讨。
1材料与方法
1.1药品与试剂鸡卵清蛋白(OVA)Ⅴ级(美国GBICO公司);氢氧化铝干粉(郑州派尼化学试剂公司);PBS(美国GBICO公司);SDF-1兔抗小鼠一抗(武汉博士德生物有限公司);过氧化物酶标记羊抗兔二抗(美国DAKO公司);ECL发光液(北京碧云天公司);兔抗小鼠内参β-actin(北京博奥森公司);免疫组织化学试剂盒(美国DAKO公司)。
1.2方法
1.2.1哮喘模型的制备小鼠哮喘模型实验参考文献[1]，略有变动。实验动物为40只雌性SPF级C57BL/6小鼠(第四军医大学动物中心提供)，体重18～22g，随机分为哮喘组和对照组。按照文献方法[1]构建哮喘模型：0、7、14d于小鼠腹腔内注射致敏液0.2mL/只(致敏液为每0.2mL PBS溶液内含10μg OVA、20μg氢氧化铝)。于第21天开始0.5g/L浓度的OVA生理盐水溶液雾化，每次30min，隔日1次，共计18次。对照组用空白PBS及生理盐水，余同模型组。两组动物于雾化结束后24h处死，取右肺于40g/L甲醛溶液中固定，石蜡包埋，用于病理观察及免疫组织化学(IH)检测;取左肺于液氮中保存，用于RT-PCR及Western-blot测定SDF-1。
1.2.2RT-PCR法检测SDF-1的表达提取肺组织总RNA，分光光度仪上定量及检测RNA纯度，用两步法RT-PCR。SDF-1上游引物：5′-CACTTTC-ACTCTCGGTCCAC-3′，下游引物为：5′-CTGAAG-GGCACAGTTTGGAG-3′，序列长度约500bp。PCR反应条件：94℃变性30s，57℃退火30s，72℃延伸1min，30个循环。RT-PCR产物以10g/L琼脂糖凝胶电泳，电泳结果在GDS-8000数码成像及分析系统上观察、拍照。