纤维粘连蛋白在哮喘气道平滑肌细胞免疫功能调控中的作用

Fibronectin Regulates Immune Function of Airway Smooth Muscle Cells in Asthmatic Rats

目的探讨细胞外基质(extracellular matrix,ECM)纤维粘连蛋白(fibronectin,FN)在哮喘气道平滑肌细胞(airway smooth muscle cell,ASMC)免疫功能调控机制中的作用,同时建立一种优化培养ASMC的方法。方法利用组织贴块法和消化酶法原代培养大鼠ASMC,并用细胞形态学和免疫组化SABC染色方法鉴定;将正常和哮喘大鼠ASMC种植在涂覆有FN的和空白(无FN涂覆)的玻片上,免疫组化方法检测爬片中粘着斑蛋白的表达;ELISA方法检测各组培养上清中调节激活正常T细胞表达和分泌细胞因子(regulated on activated normal T expressed and secreted,RANTES)蛋白水平。结果①原代培养的细胞呈典型的"谷峰状"生长,胞质内特异性的平滑肌肌动蛋白阳性表达,符合平滑肌细胞的形态学特征和生物学特性。②FN作用的哮喘大鼠ASMC粘着斑蛋白的表达明显增加,但未经FN作用的哮喘大鼠ASMC及经(或未经)FN作用的正常大鼠ASMC粘着斑蛋白表达均呈阴性表现。③FN处理组细胞培养上清中的RANTES蛋白水平较未经FN处理组显著增加(P<0.05),哮喘组培养上清中的RANTES蛋白水平较正常组明显增加(P<0.05)。结论 FN可能通过上调哮喘大鼠ASMC粘着斑蛋白的表达、促进RANTES的分泌而参与ASMC的免疫功能调控机制。

Objective To investigate the regulatory effect of fibronectin（FN）on the immune function of airway smooth muscle cell（ASMC）in asthmatic rats and to optimize the primary culture condition for ASMC.Methods Primary ASMC were cultured by digestion and tissue adherent.ASMC were identified by light microscopy and immunohistochemistry.Non-asthmatic ASMC and asthmatic ASMC were inoculated on the blank plates or the plates coated with FN.The expression of focal adhesion protein in the cell climbing film was detected by using immunohistochemistry.Regulated on activated normal T expressed and secreted（RANTES） protein in the culture supernatants was detected by using enzyme-linked immunosorbent assay（ELISA）.Results Under the light microscopy,ASMC of tracheal or bronchial origin appeared flattened and ribbon or spindle-shaped,and the expression of smooth muscle actin dispersed the cytoplasm,which was consistent with the morphological and biological characteristics of smooth muscle cells.Compared with the normal ASMC,the expression levels of RANTES protein in the culture supernatant of asthmatic ASMC were significantly increased（P0.05）.The expression levels of RANTES protein in the ASMC culture supernatants were significantly increased in the FN group（P0.05）.Conclusion FN may play an important role in the immune function of the asthmatic ASMC probably by up-regulating the expression of focal adhesion protein and promoting the secretion of RANTES protein.