摘要
目的探索分离、培养大鼠胸主动脉血管内皮细胞的有效方法。方法取Wistar大鼠1只,无菌条件下打开胸腔取出胸主动脉,去除外周结缔组织及脂肪,使主动脉内膜翻出,用丝线结扎动脉两端后用烧热的镊子烫烙。置于50ml铺有鼠尾胶培养瓶中培养,加入含有20%新生牛血清的DMEM/F12培养液静止培养,6d后换液并弃主动脉。经0.125%胰酶差速消化传代,采用免疫细胞化学法鉴定血管内皮细胞标志分子Ⅷ因子在细胞中的表达情况。结果培养6d时有少量细胞自主动脉迁移出并贴壁生长;12～14d时贴壁细胞覆盖大部分培养瓶底,约70%的细胞汇合成单层;经胰酶差速消化传代细胞生长旺盛,细胞汇合后呈现典型的"鹅卵石"样内皮细胞特征;内皮细胞标志物Ⅷ因子表达阳性率达100%。结论本研究采用的方法简便易行,不需要胶原酶及内皮细胞生长补充物,更为经济适用,适用于细小血管内皮细胞的分离与培养。
Objective To explore an effective method of isolating and culturing the vascular endothelial cells of rat thoracic aorta.MethodThe thoracic aorta was harvested under aseptic condition from the thorax of a Wistar rat.The peripheral connective tissue and fat of the thoracic aorta were stripped and disposed,and then the thoracic aorta was turned inside out to expose the intima.The thoracic aorta was ligated with silk and cauterized on both ends with heated forceps.Then the thoracic aorta was cultured in medium DMEM/F12 containing 20% newborn calf serum in a 50 ml culture bottle which was already blanketed with rat tail collagen.The thoracic aorta was discarded and the new culture medium was added into the culture bottle six days later.The migrating cells were differentially digested by 0.125% pancreatic enzyme for serial subcultivation.The cells were identified by immunohistochemical method with anti-Ⅷ factor antibody.ResultA small amount of cells were seen to migrate from the aorta and adhered to the bottom of culture bottle 6 days after cultivation;the migrating cells spread to cover most part of the bottom of culture bottle 12-14 days later.About 70% of the migrated cells were in a confluent monolayer.The confluent cells grew rapidly after being digested with pancreatic enzyme,and they showed a typical cobblestone appearance.The cells were identified as endothelial cells with 100% expression of Ⅷ factor,which was regarded as the marker of endothelial cells.ConclusionThe method established in the present study is simple and easy to handle,it does not need collagen enzyme and endothelial cell growth promoting substrate,and it is economical and applicable.It is especially suitable for isolation and cultivation of vascular endothelial cells of vessels of small caliber.
出处
《解放军医学杂志》
CAS
CSCD
北大核心
2010年第2期195-196,200,共3页
Medical Journal of Chinese People's Liberation Army
基金
吉林省科技发展计划项目(20050914)
