胰酶联合Ⅱ型胶原酶法体外分离培养软骨细胞的分析  

Primary culture of chondrocyte by trypsogen combined with type-Ⅱ collagenase

作　　者：陈林松 史宏灿 

机构地区：[1]扬州大学临床医学院胸心外科,225001

出　　处：《中国医药》2014年第10期1522-1525,共4页China Medicine

基　　金：国家自然科学基金（81170014、81370118）

摘　　要：目的 通过原代软骨细胞的体外培养和鉴定,探讨胰酶联合Ⅱ型胶原酶法体外分离培养软骨细胞的可行性.方法 分离出兔鼻中隔软骨组织,用胰酶联合Ⅱ型胶原酶的方法进行消化,获取原代软骨细胞.使用倒置显微镜观察软骨细胞的形态及生长情况,并用甲苯胺蓝染色、Ⅱ型胶原免疫组化及免疫荧光染色进行表型鉴定.结果 软骨细胞原代培养形成单层细胞需要7～8d,传代培养时间约2～3 d,细胞以圆形或类上皮细胞形态为主,甲苯胺蓝染色证实细胞可特异性合成糖胺聚糖,Ⅱ型胶原免疫组织化学染色及免疫荧光染色可证实细胞可特异性分泌Ⅱ型胶原.结论 本研究成功建立了简单易行的胰酶联合Ⅱ型胶原酶法体外分离培养大量纯净的软骨细胞,提高了消化速率,加大了细胞释放率,为组织工程气管软骨种子细胞的获取提供重要的技术支撑.Objective To explore the feasibility of seeding cells in tissue engineering trachea through primary culture and identification of cartilage cells in vitro.Methods The primary cartilage cells were isolated from the choelroseptum of rabbit,and digested by using trypsogen associated with type-Ⅱ collagenase.The morphology and growth of cartilage cells were observed by inverted microscope,and identificated by the use of toluidine blue staining,the immunohistochemistry and immunofluorescence of type-Ⅱ collagen.Results The time of form monolayer cells was 7-8 days and subculture was 2-3 days.The morphology of cartilage cells mainly consisted of circular and epithelioid cells.The cell-specific glycosaminoglycan was confirmed by the use of the method of the toluidine blue staining.The cell-specific secrete type-Ⅱ collagen was confirmed by using the immunohistochemistry and the immunofluorescence of type-Ⅱ collagen.Conclusions This study successfully establishes a simple method for isolating and cultivating chondrocytes by using the trypsinogen and the type-Ⅱ collagenase.This method may improve the digestion rate and increase the cells release rate to provide the important technical support for tissue engineered trachea seed cells.

关 键 词：软骨细胞 气管 原代细胞培养 体外生长 

分 类 号：R318[医药卫生—生物医学工程]