大鼠半月板纤维软骨细胞的分离、培养与鉴定  

Isolation,culture and identification of rats meniscal fibrochondrocytes

作　　者：杨军英[1] 林金艳[1] 韩菲 YANG Jun-ying;LIN Jin-yan;HAN Fei(College of Physical Education,Henan Normal University,Xinxiang 453007,Henan,China)

机构地区：[1]河南师范大学体育学院,河南新乡453007

出　　处：《西北师范大学学报（自然科学版）》2018年第5期68-72,共5页Journal of Northwest Normal University(Natural Science)

基　　金：新乡市科技发展规划资助项目(15SF04)

摘　　要：探讨体外大鼠半月板纤维软骨细胞分离培养与鉴定的实验方法,观察不同代次间细胞的形态学特征.手术分离大鼠双膝内外侧半月板,采用胰酶和Ⅱ型胶原酶消化法提取细胞,甲苯胺蓝染色和Ⅱ型胶原免疫荧光法鉴定细胞,低糖DMEM完全培养液常规培养.结果表明,原代半月板细胞形态呈多角形,轮廓清晰,8～10d可铺满瓶底;传代后的1～3代细胞,其贴壁及增殖能力加强,生长状态良好;传至4～5代尤其是第5代之后,细胞表型不稳定,密度稀疏,形态以长梭形为主且不规则,清晰度下降,传代周期延长;细胞鉴定结果呈阳性.双酶消化和低糖DMEM完全培养液常规培养可以获得具有良好生物学特性的半月板细胞,第4代之前的半月板细胞可以为运动损伤的组织工程学研究提供种子细胞.To discuss the method of isolation,culture and identification of rats meniscal fibrochondrocytes in vitro,observing the morphological characteristics of meniscus cells in different passages is studied.The inside and outside rats meniscus of both knees is isolated through the operation,then the meniscus cells are obtained by mechanical,enzyme and collagenase digestion methods.The cells are identified by toluidine blue staining and typeⅡcollagen immuno histochemistry staining,and then cultured normally by complete low-glucose DMEM medium.The cell can spread out fully at 8-10 d for original generation with regular morphous and clear outlinein spindle-based.The adherence and growth of meniscus cells grew rapidly from the first to the third passage and show active proliferative capacity with good conditions.The density from fourth to fifth passage,especially after fifth,is sparse and scattered.Besides,the phenotypic is instability with irregular shape,and the gap between cells are increased with lower definition.In blue staining and immunofluorescence studiesthe cells show strongpositive.The meniscus cells with good biological characteristics can be obtained by enzyme,collagenase digestion and complete low-glucose DMEM medium culture.The meniscus cells before the fourth passage maintained their biological characteristics and thus can be used as seed cells in tissue engineering about sports meniscus injury.

关 键 词：半月板 纤维软骨细胞 体外培养 鉴定 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]