人脐静脉内皮细胞培养方法的改进  

An improved culture method for human umbilical vein endothelial cells

作　　者：林敏华[1] 陈子春[1] 林宇涵[1] 许双临[1] 富显果[1] 

机构地区：[1]福建中医药大学附属宁德市医院,福建宁德352100

出　　处：《现代预防医学》2013年第24期4529-4533,共5页Modern Preventive Medicine

基　　金：福建省卫生厅青年科研课题(2011-1-45);宁德市科学技术计划项目(20110008);细胞分子生物学实验室(一级)

摘　　要：目的建立高效、可靠、经济的人脐静脉内皮细胞原代培养方法，为体外研究血管内皮细胞提供实验手段。方法取健康剖腹产新生婴儿脐带，立即用0．1％I型胶原酶室温消化，得血管内皮细胞，置于5％CO2、37℃培养箱进行原代培养，培养基采用M199培养基，含有10％胎牛血清、2mmol／L谷氨酰胺、50μg/ml肝素钠、10u／mlbFGF（贝复济，牛碱性成纤维细胞生长因子，basicfibroblastgrowthfactor）、100u／ml青霉素、50u／ml庆大霉素。在倒置显微镜下观察原代及传代细胞的形态特点，同时用免疫组织化学的方法对所得细胞进行鉴定。结果采用0．1％I型胶原酶灌注法及改良培养基获得了相当数量的人脐静脉内皮细胞。光镜下观察见大量3-4个成团的内皮细胞。细胞接种后4h开始贴壁生长，5d左右可融合成片，原代细胞呈小三角形、圆形或梭形，细胞分布不均匀，成簇生长，互不重叠；传代细胞多为扁平多角形铺路石状镶嵌排列，3d左右可融合成片。免疫组化可见内皮细胞胞浆中人第Ⅷ因子相关抗原呈阳性反应。结论I型胶原酶灌注法是获得脐静脉内皮细胞的一种可取方法，成功率高，可靠性大。改良培养基能很好地促进内皮细胞体外培养及传代扩增，可成功构建体外研究血管内皮细胞的模型。OBJECTIVE The study aimed to establish an efficient, reliable, and economical method for culturing primary human umbilical vein endothelial cells （HUVECs） , so to provide an experimental approach for in vitro studies on vascular endothelial cells. METHODS Umbilical cords of healthy C-sectioned newborn infants were removed and digested with 0.1% collagenase I at room temperature to obtain vascular endothelial cells. The cells were incubated under 5% CO2 at 37~C and primary-cultured in M199 culture medium containing 10% fetal bovine serum, 2 mmol/L of glutamine, 50 p,g/ml of heparin sodium, 10 u/mlb of FGF （basic fibro blast growth factor）, 100 μ/ml of penicillin, and 50 μ/ml gentamicin. Morphological features of the primary and passaged ceils were observed under an inverted microscope, and were identified by immunohisto- chemical methods. RESULTS A considerable amount of HUVECs were obtained by perfusion with 0.1% collagenase I and by using the improved culture medium. Large amount of cell clusters comprised of 3-4 endothelial cells were observed under an optical microscope. The cells began to adhere to the bottom of the flasks 4 h after seeding and reached confluency on around day 5. The primary cells were small triangular, round, or spindle in shape, distributed unevenly, and grew in non-over- lapped clusters. On the other hand, the passaged cells were arranged in a flat and polygonal paving stone-like pattern, and the cells could reach confluency in about 3 days. Immunohistochemical analysis revealed that cytoplasm of the endothelial cells contained human factor VIII related-antigens. CONCLUSION Collagenase I perfusion is a feasible method for obtaining HU- VECs; it is high in success rate and reliability. The improved medium is useful in promoting culturing of endothelial cells in vitro and proliferation of passaged cells, and has demonstrated to successfully establish in vitro models for studies on vascular endothelial cells.

关 键 词：人脐静脉内皮细胞 鉴定 细胞培养 

分 类 号：R329-33[医药卫生—人体解剖和组织胚胎学]