胶原酶脐带灌注消化法在人脐带静脉内皮细胞的原代分离培养中的作用

Application of collagenase perfusion and digestion techniques in the primary culture of endothelial cells from human umbilical vein in vitro

目的探讨人脐带静脉内皮细胞原代分离培养方法,为血管内皮功能及其相关疾病的研究奠定基础。方法采用0.1%Ⅰ型胶原酶脐带静脉灌注消化法,分离得到血管内皮细胞后,加入M199混合培养液,置于37℃、5%CO2培养箱中培养,以0.25%胰蛋白酶进行消化传代培养。细胞鉴定采用形态学方法、透射电镜法以及细胞Ⅷ因子免疫化学法。结果原代人脐静脉内皮细胞约24h后完全贴壁,第3～4天融合成单层,呈铺路石状镶嵌排列;细胞透射电镜观察可见特征性Weibel-Palade小体以及细胞表面大量微绒毛;细胞Ⅷ因子抗原免疫组化以及荧光显示均呈阳性反应,证实所分离培养细胞为人脐带静脉内皮细胞。结论采用胶原酶脐带静脉灌注消化法可分离得到较为纯化的内皮细胞,并可在体外连续大量增殖传代,5代内的细胞均可用于研究。

Objective To investigate the primary culture method for the human umbilical vein endothelial cells (HUVECs). Methods Endothelial cells in human umbilical vein were got by the method of 0.1 % Ⅰtype collagenase perfusion and digestion techniques for about 10 min,and then cultured with M199 mixed culture medium in Incubator with 5% CO2 at 37 ℃,0.25% trypsin were used for cell transfer of culture. The cultured endothelial cells were identified by cell morphology,cell micro-structure,Ⅷ factor antigen immunohistochemitry. Results The primary cultured HUVECs could attached to flask completely after 24 hours,which arrayed like pitching stone under microscopy.By transmission electron microscopy,HUVECs were found to have distinctive Weibel-Palade bodies in the cytoplasm and a lot of microvilli in the cell surface. Immunohistochemitry and immmunofluorescent staining by Ⅷ factor were all positive reaction. All of these were the typical features of primary cultured HUVECs. Within five generations,the morphology of cells was not obviously changed. Conclusions High purity HUVECs can be obtained by the technique of collagenase perfusion and digestion,which further can be passaged successfully in vitro,and cells within five generations can be used in research.