论文部分内容阅读
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 study the optimal conditions of human umbilical vein endothelial cells (HUVECs) for starvation, to establish an efficient and stable method for the isolation and purification of HUVECs in vitro and to provide the basis for HUVECs-related experiments. Methods Human umbilical vein was digested with 0.1% collagenase for 15 min. The cells were harvested and cultured with endothelial cell specific medium (containing 5% endothelial cell basal medium, 1% endothelial cell growth factor, 1% streptomycin) 37 ℃, 5% CO2 incubator. The morphology of the cells was observed under an inverted microscope, and the cells were identified by immunofluorescence. The purity of HUVECs was detected by flow cytometry. The effects of 0, 0.1, 0.5% and 1% serum concentration on the cell cycle were evaluated by starvation at 0, 6, 12, 18 and 24 h. Results HUVECs were obtained by 0.1% collagenase digestion. The cells were cultured in a typical cobblestone arrangement, and the cells were arranged in a spiral pattern. Immunofluorescence assay showed that the expression of related factor VIII antigen was positive. Flow cytometry purity of up to 99.67%. About 70% G0 / G1 phase cells were obtained after cultured with different concentrations of serum for 6 h, 80% -90% G0 / G1 phase cells were obtained after cultured for 12 h, 95% G0 / G1 phase cells. Conclusion 0.1% type Ⅱ collagenase filled the vein lumen can obtain high purity primary HUVECs. The G0 / G1 phase HUVECs with the purity of more than 80% can be obtained by completely serum-free medium for 12 h.