摘要
目的建立一种高效分离、培养大鼠胸主动脉血管内皮细胞的方法。方法用Wistar大鼠(200～250 g),无菌条件下开胸取胸主动脉,去除血管外膜,制备长约1.0～1.5 mm动脉环。将血管环垂直置于干燥的培养皿中,并向血管环内加入含有10%胎牛血清的DMEM/F12培养液、静止培养;24 h血管环贴壁后,加入培养液;3～4 d内皮细胞长出,弃血管环,并经0.25%胰蛋白酶消化传代。用形态学、免疫细胞化学及流式细胞分析等方法,鉴定血管内皮细胞标志物,vWF的表达。结果培养3～4 d时有细胞自主动脉环内迁移出并贴壁生长,细胞汇合后呈现典型的"铺路石"样内皮细胞特征;免疫组化和流式细胞分析结果示,vWF表达阳性率可高达98.3%±0.2%。结论用改良的植块培养方法,不需要胶原及内皮细胞生长补充物,较传统酶消化法更为经济、高效,且简便易行,适用于细小血管内皮细胞的分离与培养。
Objective To establish a simple and efficient method for isolating and culturing vascular endothelial cells of rat thoracic aorta.Methods Under aseptic condition,the thoracic aorta was separated from Wistar rat.After stripping off the adventitia,the thoracic aorta was cut into vessel rings with length of 1.0～1.5 mm.The vessel rings were placed on culture dish vertically and filled with the culture medium DMEM/F12 containing 10% fetal bovine serum;After 24 h,the vessel rings had attached on the culture dish firmly and some new culture medium was added;3～4 d later,the thoracic aorta was discarded and the new culture medium was added into the culture dish.The migrating cells were digested by 0.25% trypsin for serial subcultivation.The endothelial cells were identified by morphological,immunohistochemical and flow-cytometry methods with anti-vWF antibody.Results A amount of cells migrated from the aorta and adhered to the bottom of culture dish 3～4 d after cultivation.The confluent cells grew rapidly after being digested with 0.25% trypsin and showed the typical cobblestone appearance.The cells were identified as endothelial cells,its positive ratio was（98.3±0.2）% by using immunostaining and flow-cytometry with vWF.Conclusion The modified explanting cultivation method for isolating and culturing was established in this study.This method is simple,easy to handle and more economic without need of collagen and endothelial cell growth promoting substrate.
出处
《基础医学与临床》
CSCD
北大核心
2011年第11期1278-1282,共5页
Basic and Clinical Medicine
基金
国家自然科学基金(30871219)
国家重点基础性研究项目(2006CB5041)
北京市教育委员会科技计划重点项目(KZ200810025012)
北京市属高等院校人才强教计划项目(京教人[2008]17号)
北京市新世纪百千万人才工程培养经费(08-016)
教育部高等学校博士点科研基金(20091107110001)
关键词
内皮细胞
胸主动脉
大鼠
endothelial cells
thoracic aorta
rats
