鸡胚肺动脉内皮细胞与平滑肌细胞的分离培养及鉴定  

Cultivation and identification of the chick embryo pulmonary artery endothelial cells and smooth muscle cells

作　　者：韩燕燕[1,2] 刘思当[1] 杨鹰[2] 

机构地区：[1]山东农业大学动物科技学院,山东泰安271000 [2]中国农业大学动物科学技术学院,北京100193

出　　处：《中国农业大学学报》2009年第2期17-21,共5页Journal of China Agricultural University

基　　金：国家“十一五”支撑计划(2006BAD12BO7-4);中国农业大学科研启动基金(2006001)

摘　　要：探索简单快速实用的鸡胚肺动脉内皮细胞和平滑肌细胞培养方法：在严格无菌条件下对肺动脉内皮细胞和平滑肌细胞分离培养，待细胞融合度达约80％时进行传代，得到的内皮细胞利用差速消化法纯化，并观察2种细胞形态特征及培养特性，待细胞稳定生长后，采用荧光免疫组化技术鉴定细胞，并进行冻存和复苏，观察复苏后细胞活性。结果显示：24h后组织块周围即有少量细胞爬出，内皮细胞大量生长后呈突起状排列，立体感强，平滑肌细胞在培养后的第5天呈现“峰谷”样外观，经免疫荧光法证明是内皮细胞和平滑肌细胞的纯培养，冻存于液氮中1～2个月复苏后细胞的活性都较好，可以恢复旺盛生长。结论：本研究提出的组织贴块法能成功获得内皮细胞和平滑肌细胞的纯培养，比酶消化法操作简单，更适合小血管内皮细胞、平滑肌细胞的原代培养。To explore the fast and feasible method of cultivating endothelial cells （ECs） and smooth muscle cells （SMCs） from the pulmonary artery in chick embryo. Isolating and cultivating the pulmonary artery endothelial cells （PAECs） and the smooth muscle cells （PASMCs） from the chick embryo by tissue-sticking methods under the strictly sterile condition. The PAECs were sublimated after passaged when the monolayer confluence reached 80%. Two different morphologic characteristics and cultural character of the cells were observed till the cells growth stabilized. The ECs were characterised by positive immunofluorescent staining for von Willebrand Factor and the SMCs were characterized by positive immunofluorescent staining for α-SMCs-action. The cytoactive after resuscitation was observed. The results showed that few cultivated cells were found after 24h, and the subcultured ECs grown on the treated dish showed cobblestone after sublimated and have great three dimension appearance. The PASMCs had developed into groups like peak-valley shape after cultivated 5d. The cells were identified as pulmonary artery endothelial cells and smooth muscle cells by immunofluorescence assay respectively. All the cells could grow productively after resuscitation. A reliable method is introduced to isolate and propagate the endothelial cells and vascular smooth muscle cells from pulmonary artery of chicken by cultivating small tissue cube. This method is simpler than enzyme digestion method, and more suitable compared with the method of primary cultivation of small pulmonary artery endothelial cells and smooth muscle cells.

关 键 词：鸡胚 肺动脉 内皮细胞 平滑肌细胞 培养 免疫组化 鉴定 

分 类 号：S852.2[农业科学—基础兽医学]