大鼠沃勒变性面神经施万细胞纯化培养实验研究  

PURIFICATION OF CULTURED SCHWANN CELLS DERIVED FROM WALLERIAN DEGENERATING FACIAL NERVES IN RATS

作　　者：马训[1] 俞光岩[1] 张震康[1] 李盛琳[1] 傅嘉[1] 章魁华[1] 

机构地区：[1]北京大学口腔医学院口腔颌面外科,北京100081

出　　处：《解剖学报》2001年第3期205-208,T002,共5页Acta Anatomica Sinica

基　　金：国家自然科学基金资助课题 (3 95 70 765 )

摘　　要：目的　研究大鼠沃勒变性段面神经施万细胞 (SchwanncellsfromWalleriandegeneratingfacialnerve ,WFSC)分离纯化、培养方法 ,观察其形态表型和一般生物学特性。　方法　采用“双差速粘附”法纯化大鼠WFSC ,以抗S 10 0蛋白、抗p75 LNGFR 和抗纤维粘连蛋白 (抗FN)抗体免疫细胞化学染色和改良Masson染色进行鉴定 ;分析比较计数法和MTT测定法细胞生长规律 ;观察传代培养和冷冻复苏的细胞形态和生长特征。　结果　免疫细胞化学染色抗S 10 0蛋白阳性WFSC >90 % ,抗p75 LNGFR阳性WFSC >82 % ,抗FN阳性WFSC <5 %～ 7% ;绝大部分WFSC改良Masson特染阴性。传代培养和冷冻复苏后的细胞形态表型及生长特性与原代细胞基本相同 ,皆呈双极梭形 ,指数增殖期在培养接种后 4～ 6d ,体外生长呈有限增殖和明显的接触抑制性。　结论　采用“双差速粘附”法可获得较高纯度面神经施万细胞 ;沃勒变性面神经施万细胞可能在体内创伤微环境及多种因子作用下 ,具备了活跃的生长、增殖能力 ,在体外培养条件下 ,其形态表型、生长规律等生物学特性与文献报道的其他外周神经施万细胞具有相似性。Objective To investigate techniques among isolation,purification,proliferation of Schwann cells derived from Wallerian degenerating facial nerve(WFSC) in rats and to study the characteristics in morphological phenotype and growth in vitro. Methods By means of modified “differential adhesion”,the cultivated cells of WFSC were identified by using immunocytochemical stain with antibodies to S\|100 protein,p75 LNGFR and fibronectin and specific staining of Masson's modification.Growth feature of WFSC in vitro was estimated depending on cell counting and MTT assay. Results Cultured cells predominantly showed spindle bipolar shape with purity of typical WFSCs more than 90% in anti\|S\|100 positive and more than 82% in anti\|p75 LNGFR positive individually.The exponential growth was within 4th to 6th day after inoculation with obvious limited reproduction and contact inhibition. Conclusion Higher purity of WFSCs might be obtained with modified “differential adhesion” purification and that their active growth and reproduction in vitro was probably due to tropic stimulation by many cytokines around the trunk of injured facial nerve in vivo and by the stimulation of autocrined growth promoting factors or in NBS.The biological characteristics of them might be similar to Schwann cells from other regions of peripheral nerve system.The results demonstrated that cultured WFSCs could be used as an ideal cell model for investigating the trophic effect and its mechanism of promoting regeneration of injured facial nerve. [

关 键 词：施万细胞 沃勒变性 免疫细胞化学 面神经 细胞培养 大鼠 

分 类 号：R745.12[医药卫生—神经病学与精神病学]