摘要
目的探讨人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs)的最佳饥饿条件,以建立高效稳定的HUVECs体外分离提取方法,并为HUVECs相关实验提供研究基础。方法用0.1%Ⅱ型胶原酶灌注脐静脉腔消化15 min,收集细胞并用内皮细胞专用培养基(含5%内皮细胞基础培养基、1%内皮细胞生长因子、1%青链霉素),置37℃,5%CO2培养箱培养。在倒置显微镜下观察细胞形态特点,并用细胞免疫荧光方法对所得细胞进行鉴定。用流式细胞术检测所得HUVECs的纯度,并检测0%、0.1%、0.5%、1%血清浓度的培养基饥饿处理0、6、12、18、24 h对细胞周期的影响。结果 0.1%Ⅱ型胶原酶消化可以得到HUVECs,细胞培养呈典型的铺卵石状排列,细胞较密处呈涡旋状排列。免疫荧光检测细胞VIII因子相关抗原表达呈阳性。流式检测细胞纯度高达99.67%。不同血清浓度培养基培养6 h可获得70%左右的G0/G1期细胞;培养12 h可获得80%~90%的G0/G1期细胞;培养18、24 h可获得95%左右的G0/G1期细胞。结论 0.1%Ⅱ型胶原酶充盈静脉管腔可以获得高纯度的原代HUVECs。完全无血清培养基培养12 h即可获得纯度超过80%的G0/G1期HUVECs。
Objective To investigate the optimal starvation conditions of human umbilical vein endothelial cells(HUVECs) and establish a highly efficient and stable method for separating HUVECs. Methods HUVECs harvested from human umbilical cords by digestion with 0.1% collagenase II for 15 min were cultured in endothelial culture medium(ECM) containing 5% fetal bovine serum(FBS), 1% endothelial cell growth factor(ECGS) and 1% penicillin/streptomycin solution(P/S) at 37 ℃ in 5% CO_2.The cells were observed for cell morphology under an inverted microscope and identified with immunofluorescence assay. The purity of HUVECs was detected using flow cytometry(FCM). The cell cycles of HUVECs cultured in the presence of 0, 0.1%,0.5%, and 1% FBS for 0, 6, 12, 18, and 24 h were analyzed with flow cytometry. Results The purity of HUVECs harvested by digestion with 0.1% collagenase II reached 99.67%. The primary HUVECs showed a cobblestone or volute appearance in vitro.Immunocytochemistry showed that HUVECs highly expressed VIII-related antigen. Cell culture in the presence of different concentrations of FBS for 6 h resulted in 70% G_0/G_1 phase cells, which increased to 80%-90% at 12 h of cell culture, and further to around 95% at 18 and 24 h. Conclusion Digestion with 0.1% collagenase II can obtain high-purity primary HUVECs.Culturing HUVECs in serum-free medium for 12 h can result in a high purity(over 80%) of G_0/G_1 phase cells.
出处
《南方医科大学学报》
CAS
CSCD
北大核心
2016年第8期1140-1143,共4页
Journal of Southern Medical University
基金
国家自然科学基金(81370449)
广州市科技计划项目产学研协同创新重大专项(201504291720212)~~
关键词
人脐静脉内皮细胞
细胞原代培养
血清饥饿
细胞周期
human umbilical vein endothelial cells
primary culture
serum starvation
cell cycle
