人脐静脉内皮细胞的体外分离培养及鉴定

Cultivation and in vitro identification of endothelial cells from human umbilical vein

目的探讨人脐静脉内皮细胞的原代培养方法,提高体外分离培养血管内皮细胞的成功率。建立血管内皮细胞培养模型,为体外研究血管内皮细胞提供实验基础。方法取2根脐带(至少20 cm)冲净淤血,采用加工穿刺针固定脐静脉灌注消化液,一根用0.2%胶原酶Ⅱ,另一根用0.1%胶原酶Ⅰ和0.25%胰酶等比混合消化液,比较两种酶的消化效果。收集细胞并用含有10 ng/mL的VEGF的培养基中培养,观察细胞的生长及传代。并在倒置显微镜下观察细胞的形态学特点,同时用免疫组织化学的方法对所得细胞进行鉴定。用流式细胞术观察细胞周期。结果两种消化酶方法均获得了相当数量的人脐静脉内皮细胞,胶原酶Ⅱ的消化效果稍优于混合消化酶,且比较理想的消化时间均为13 min,细胞接种后4～5 h开始贴壁生长,1周左右可生长成单层,光镜下呈多角形,"铺路石"样排列,免疫组织化学法可见内皮细胞胞浆中人第Ⅷ因子相关抗原呈阳性反应。细胞周期显示约有50.6%的细胞处于G0/G1期。结论胶原酶Ⅱ和胶原酶Ⅰ与胰酶等比混合消化液灌注法是获得脐静脉内皮细胞的一种可取方法,而胶原酶是分离培养脐静脉内皮细胞的首选消化液,成功率高,可靠性大,可成功构建体外研究血管内皮细胞的模型。

Objective To explore the primary culture method of human vascular endothelial cells（ECs） from umbilical vein, enhance the success rate of separating and culturing ECs in vitro, establish the model of human vascular ECs, and provide an experimental method for research of ECs. Methods The umbilical veins in human umbilical cords, 20 cm long, were fixed and filled the digestive fluids, 0.2% collagenenase Ⅱ in one cord and the complex enzyme of 0.25% trypsin and 0.1% collagenase Ⅰ in an other, and the digestive results were compared. All the cells were collected and cultured in fluid with 10 mg/mL VEGF, their procreation and generation were observed, the morphologic characteristics of ECs were observed with light microscopy and immunohistochemistry under inverted microscope, aud the cell cycle was observed by flow cytometer. Results Enough ECs were obtained from both digestive liquids, and collagenenase Ⅱ was better than complex enzyme, the effective digesting time was 13 minutes, the culturing cells grew on the wall appeared after 4-5 hours and the monolayer cells were formed ,after one week. Ⅷ factor in the ECs showed positive reaction by immunohistochemistry. Cell cycle revealed that 50.6% cells were at stage of G0/G1. Conclusion The perfusion with collagenenase Ⅱ and the complex enzyme and collagcnase Ⅰ is an effective method to gain ECs, and collagenenase is the digestive fluid for primary choice, and can successfully establish the model for research of ECs.