豚鼠鼻黏膜成纤维细胞的分离、培养及鉴定

Isolation,culture and identification of guinea pig nasal mucosa fibroblasts

背景:鼻黏膜成纤维细胞可通过分泌多种细胞因子和趋化因子,参与多种鼻部疾病的炎症反应和损伤修复;豚鼠是最适合用于变态反应性疾病研究的实验动物之一。目的:探讨豚鼠鼻黏膜成纤维细胞分离、培养、纯化和鉴定的有效方法。方法:胶原酶消化法分离豚鼠鼻黏膜成纤维细胞,经差速贴壁法纯化后,采用倒置相差显微镜进行形态学观察,波形蛋白免疫细胞化学法鉴定成纤维细胞,并通过MTT检测法绘制细胞生长曲线。结果与结论:①倒置相差显微镜下可见原代鼻黏膜成纤维细胞中有不同形态细胞交杂生长,以类梭形和扁平状细胞为主,细胞成团散落分布;纯化后的成纤维细胞形态均一,多呈长梭形,分布较均匀,呈鱼群状或放射状排列;②免疫细胞化学染色显示,成纤维细胞波形蛋白表达呈阳性;③细胞生长曲线大致呈"S"形。④结果表明,所分离培养的细胞具有典型的成纤维细胞生物学特征。

BACKGROUND:Nasal mucosa fibroblasts are reported to involve in inflammation and wound repair of various nasal diseases by secreting a variety of cytokines and chemokines.Guinea pig is a most suitable experimental animal for the study of allergic diseases.OBJECTIVE:To investigate the effective methods of isolation,culture,purification and identification of guinea pig nasal mucosa fibroblasts.METHODS:Guinea pig nasal mucosa fibroblasts were isolated by collagenase digestion,and purified by differential velocity adherence.The morphology of fibroblasts was observed by inverted phase contrast microscope,fibroblasts were identified by vimentin immunocytochemical staining,and the cell growth was detected by MTT assay to draw the growth curve.RESULTS AND CONCLUSION:(1)Under inverted phase contrast microscope,there were cells with different shapes in the primary nasal mucosa fibroblasts,most of which showed spindle-like and applanate shape,and cells were scattered distribution in cluster.The purified fibroblasts were homogeneous,mainly were long spindle-shaped,and distributed in fish shoal-like and radial-like.(2)Immunocytochemical staining indicated that fibroblasts were positive for vimentin.(3)The cell growth curve appeared to be typical S-shaped.(4)To conclude,the isolated and cultured cells exhibit typical biological characteristics of fibroblasts.