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目的:探讨Ⅰ型神经纤维瘤病(type 1 neurofibromatosis,NF-1)肿瘤组织中成纤维细胞(fibroblast,FB)体外分离、培养及鉴定的方法。方法:以4例NF-1患者的神经纤维瘤组织为材料,采用组织块法分离培养FB,细胞铺满培养瓶底后传代,差速贴壁法(15min/次)纯化P1、P2代FB。倒置显微镜观察细胞形态学变化,并用免疫细胞化学方法检测成纤维细胞特异蛋白-1、波形蛋白和S-100蛋白表达情况,对培养细胞进行鉴定。结果:组织块法原代培养约5d可见大量细胞从组织块周围迁出,20d左右达80%～90%融合。经纯化后P3代FB成纤维细胞特异蛋白-1和波形蛋白免疫组化染色阳性,阳性率分别为99.4%和99.6%;S-100染色阴性。结论:组织块培养法结合差速贴壁法可成功分离出高纯度NF-1肿瘤组织内FB,该培养方法是一种较理想的NF-1肿瘤组织FB培养方法。
Objective: To investigate the isolation, culture and identification of fibroblast (FB) in type 1 neurofibromatosis (NF-1) in vitro. Methods: The neurofibromatosis tissues of 4 patients with NF-1 were used as material. The fibroblasts were isolated and cultured by tissue block method. The cells were passaged after culturing on the bottom of culture flask. The P1 and P2 were purified by differential adherence method (15min / time) . The morphological changes of the cells were observed by inverted microscope. The expression of fibrinogen-specific protein-1, vimentin and S-100 protein was detected by immunocytochemistry, and the cultured cells were identified. Results: After cultured for about 5 days in the tissue culture method, a large number of cells migrated around the tissue mass, reaching about 80% -90% fusion around 20 days. After purification, P3 fibroblast-specific protein-1 and vimentin immunohistochemical staining were positive in P3 generation, the positive rates were 99.4% and 99.6% respectively; S-100 staining was negative. Conclusion: Tissue mass culture combined with differential adherence method can successfully separate FB from high purity NF-1 tumor tissue. This culture method is an ideal FB culture method for NF-1 tumor tissue.