人脐静脉内皮细胞原代培养方法的改进  

Human umbilical vein endothelial cells culture: improvement and experience

作　　者：高峰[1] 

机构地区：[1]福建省宁德市医院心血管内科,宁德352000

出　　处：《福建医药杂志》2013年第2期64-67,共4页Fujian Medical Journal

摘　　要：目的改进人脐静脉内皮细胞(HUVECs)原代培养方法,以获得足够数量的高纯度可传代内皮细胞。方法胶原酶消化法分离培养内皮细胞,并加以改进。2.5×104 IU/L重组牛碱性成纤维细胞生长因子(rb-bFGF)加入完全培养液中。用形态学、免疫细胞化学染色法进行内皮细胞鉴定。观察含rb-bFGF的培养液对内皮细胞生长和形态结构的影响。观察原代细胞长满瓶底60%、70%和80%时传代后第1代细胞的生长情况。结果 HUVECs融合时呈单层铺路石样外观,95%以上细胞Ⅷ因子检测呈阳性。含2.5×104 IU/L rb-bFGF的完全培养液可将HUVECs传7代以上。第7代细胞长满瓶底80%所需时间延长,并出现异常细胞形态。原代细胞长满瓶底60%、70%和80%后传代,第1代细胞长满瓶底80%所需时间均为3d左右。结论 rb-bFGF可取代内皮细胞生长添加剂、内皮细胞生长因子等添加于培养液中,获得足够数量高纯度可传代的内皮细胞;原代细胞的传代时间可适当提前至长满瓶底60%～70%时。Objective To improve the method of culturing human umbilical vein endothelial cells （HUVECs） in vitro to obtain enough qualified and high purity HUVECs. Methods The procedures of isolating, culturing, and subeulturing HU VECs were improved. The recombinant bovine basic fibroblast growth factor （rb-bFGF） （2.5 × 104 IU/L） was added to culture medium. The cultured cells were verified as HUVECs by morphology and Immunocytochemical staining. The effects of culture medium added rb-bFGF on HUVECs proliferation were investigated by continuously observing the growth and morphology of the primary cells to the seventh passage of cells with an inverted phase contrast microscope. The primary culture cells were passaged when they grew to 60%, 70%, 80% confluence, and the growth of the first passage of cells was viewed. Results The culture cells had a cobblestone appearance, and the purity of endothelial cells was more than 95% identified by Immunocytochemical staining for V~ factor. Culture medium added rb-bFGF could passage the endothelial to at least seventh passage. The first pas sage of cells grew to 80% confluence after about three days of culture, no matter the primary culture cells were passaged when spreading 60~, 70% or 80% of the whole culture area. Conclusion rb-bFGF might be a replacement for ECGS or VEGF in HUVECs culturing. The passaging time of the primary culture cells could be advanced properly.

关 键 词：人脐静脉内皮细胞 细胞培养 碱性成纤维细胞生长因子 倒置相差显微镜 

分 类 号：R331[医药卫生—人体生理学]