原代培养大鼠颌下腺细胞分离方法的比较研究  

COMPARATIVE STUDY ON THE ISOLATING METHODS OF RAT SUBMANDIBULAR GLAND CELL FOR PRIMARY CULTURE

作　　者：周青[1] 王玉新[1] 朱耀旻[1] 王绪凯[1] 卢利[1] 孙长伏[1] 

机构地区：[1]中国医科大学第一临床学院颌面外科整形外科,沈阳110001

出　　处：《中国修复重建外科杂志》2003年第2期169-172,共4页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：辽宁省科技攻关资助项目 (0 0 2 2 50 0 1 )~~

摘　　要：目的　探讨体外培养大鼠颌下腺细胞的分离方法并进行比较。方法　取 Wistar大鼠颌下腺分别采用直接分离法与胰酶消化法分离获取颌下腺细胞 ,并进行原代及传代培养 ;相差倒置显微镜观察细胞形态 ,苔盼蓝染色法测细胞成活率 ,MTT法检测培养细胞活性 ,并检测 2 4、4 8、72和 96小时不同时间培养上清液中淀粉酶的含量 ,免疫组织化学鉴定细胞来源及比例。结果　直接分离法获取的细胞活性为 70 % ,活力值为 0 .16± 0 .0 14 ,功能欠佳 ,经胰酶消化法所获取的细胞活性为 85 % ,活力值为 0 .4 5± 0 .0 13,细胞形态完整、贴壁良好 ,且功能强 ;免疫组织化学检测颌下腺细胞α- SMA抗体、CK8.13抗体、keratin抗体呈阳性反应 ,胰酶消化法颌下腺细胞反应强度大于直接分离法。结论　胰酶消化分离获取颌下腺细胞的方法优于直接分离法。Objective To explore the isolating methods of rat submandibular gland cell for primary culture. Methods Rat submandibular gland cell were isolated by direct isolation and pancreatin digestion respectively, and then were cultured and subcultured on DMEM. The shape and structure of cultured cells were observed with phase contrast microscope. The cell survival rate was detected by using trypan blue elimination test. The vital force of culture cells was estimated with MTT colorimetric method. The cultured cell secretion function was evaluated by assay of amylase activity. Results By direct isolation, the cell survival rate was 70% and the cell vital force was 0.16±0.014. By pancreatin digestion, the cell survival rate was 85% and the cell vital force was 0.45±0.013; the cells had good shape and attached well. The CK8.13 and keratin antibodies were epithelium specific and α SMA antibodies were myoepithelium specific. The cells were stained positively with CK8.13, keratin and α SMA antibodies. Conclusion The method of pancreatin digestion for the isolation of submandibular gland cell is better than that of direct isolation.

关 键 词：颌下腺细胞 分离 胰酶消化法 原代培养 传代培养 细胞形态 细胞学 

分 类 号：R318[医药卫生—生物医学工程]