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目的建立一种简便、高效的犬脐静脉血管内皮细胞分离方法,并进行培养和鉴定。方法无菌条件下取12只新生比格犬脐带共12根,脐带长(13.0±1.5)cm。PBS液冲洗后,各取4根脐带注入1%Ⅰ型胶原酶分别消化脐静脉5、7、10 min后,收集细胞并行锥虫蓝计数;倒置相差显微镜下观察细胞形态,待细胞爬满培养瓶后以1∶2比例传代。取Ⅰ型胶原酶消化7 min获得的第3代细胞,通过免疫荧光染色和流式细胞仪检测细胞中血管假性血友病因子(vonWillebrand factor,vWF)和血小板-内皮细胞黏附分子CD31表达,进行细胞鉴定;MTT检测细胞增殖。结果消化5、10 min后未收集到或偶尔收集到血管内皮细胞;消化7 min可见大量血管内皮细胞,培养24 h后细胞呈多角形,随培养时间延长细胞逐渐呈“铺路石”样生长,细胞核较大,多为单核,少数细胞为双核,传代后细胞形态无明显变化。荧光倒置显微镜下观察,培养细胞vWF和CD31表达阳性;流式细胞仪检测示培养细胞vWF阳性表达率为99.0%±0.7%,CD31阳性表达率为98.0%±1.2%,提示培养细胞为血管内皮细胞。MTT检测示,随培养时间延长,血管内皮细胞增殖速度明显提高。结论采用酶消化法从犬脐静脉中分离培养血管内皮细胞操作简便,且细胞数量多、纯度高。
Objective To establish a simple and efficient method for the isolation of canine umbilical vein endothelial cells and culture and identification. Methods A total of 12 umbilical cords from 12 newborn beagle dogs were obtained under aseptic conditions. The length of umbilical cord was (13.0 ± 1.5) cm. After washing with PBS solution, 4 umbilical cord umbilical cords were instilled into 1% type Ⅰ collagenase to digest umbilical vein respectively for 5, 7 and 10 min, then the cells were harvested for trypan blue counting. The morphology of cells was observed under inverted phase contrast microscope. After the bottle to 1: 2 ratio of passage. The third generation of cells obtained after digestion with collagenase type I for 7 min were used to detect the expression of von Willebrand factor (vWF) and platelet-endothelial cell adhesion molecule CD31 in the cells by immunofluorescence staining and flow cytometry. Cell identification; MTT detection of cell proliferation. Results After 5 and 10 min of digestion, no vascular endothelial cells were collected or occasionally collected. After digestion for 7 min, a large number of vascular endothelial cells were observed. After cultured for 24 h, the cells were polygonal, and gradually became “paving stones” Growth, larger nuclei, mostly mononuclear, a small number of dual-nucleated cells, cell morphology after passage no significant change. Fluorescence inverted microscope showed that vWF and CD31 were positive in cultured cells. Flow cytometry showed that the positive expression rate of vWF in cultured cells was 99.0% ± 0.7% and the positive expression rate of CD31 was 98.0% ± 1.2%, which indicated that the cultured cells were vascular endothelial cell. MTT test showed that with the extension of culture time, vascular endothelial cell proliferation rate was significantly increased. Conclusion Enzyme digestion method for isolation and culture of vascular endothelial cells from canine umbilical vein is simple and convenient, and the number of cells is high and purity is high.