人脐静脉内皮细胞体外培养方法的改进

Improvement of human umbilical vein endothelial cells culture in vitro

目的建立一种能大量分离人脐静脉内皮细胞(HUVECs)的体外培养方法,为血管内皮细胞的大规模体外实验研究奠定基础。方法比较0.25%胰酶、0.1%Ⅰ型胶原酶、0.25%胰酶与0.1%Ⅰ型胶原酶(1∶1)3种消化方法分离HUVECs的细胞数量和活细胞率的差异。观察血管内皮细胞生长因子(VEGF)对细胞生长的影响。细胞鉴定采用形态学观察和免疫荧光法检测。结果0.1%Ⅰ型胶原酶单独或混合0.25%胰酶消化分离细胞数量较多、活细胞率较高(P<0.05)。VEGF能明显促进细胞增殖能力,延长细胞自然生长时间。细胞呈单层"铺路石"样排列,免疫荧光细胞化学法染色,激光共聚焦检测可见胞质中绿色荧光颗粒。结论Ⅰ型胶原酶与胰酶混合消化HUVECs是获取血管内皮细胞的一种经济且效果较好的方法,添加VEGF能明显促进细胞增殖,可用于构建体外研究血管内皮细胞的模型。

Objective To establish the methods of isolating and culturing human umbilical vein endothelial eells（HUVECs） in vitro. Methods The number and viability of HUVECs respectively isolated by 0. 25% trypsinase, 0. 1% type Ⅰ collagenase, the mixture of 0.1% type i collagenase and 0.25% trypsinase（1 ： 1） were tested by cell counting and trypan blue exclusion methods, Cell growth curve drawing were used to observe the effect of vascular endothelial cell growth factor（VEGF） on the proliferation of HUVECs. HUVECs were identified by morphological observation and immunofluorescencal method, Results Both 0. 1% type I collagenase and the mixture of 0.1% type Ⅰ collagenase and 0.25% trypsinase （1 ： 1） were superior to 0.25% trypsinase both for cell number and viability. VEGF could significantly promote the proliferation of HUVECs（P〈0, 05）, HUVECs were presented as ＂slab-stone＂ arrangement, and the antigens of Ⅷ factor were positive according to the immunofluorescencal method. Conclusion Ⅰ solating by 0. 1% type Ⅰ collagenase mixtured with 0.25% trypsinase（1 ：1） and culturing by the medium with VEGF is an economical and practical way for HUVECs culture in vitro.