血管内皮细胞生长因子受体2对原代人脐静脉内皮细胞作用研究

Effects of vascular endothelial cell growth factor receptor 2 on primary human umbilical vein endothelial cells

目的研究核糖核酸(RNA)干扰血管内皮细胞生长因子受体2(VEGFR2)对原代人脐静脉内皮细胞(HUVECs)迁移及血管生成的影响。方法从新生儿脐带中提取原代HUVECs。将细胞分为3组:空白组、阴性对照组和实验组。空白组细胞不做任何处理,阴性对照组加入100 nmol·L^-1人与小鼠基因无同源性的小干扰RNA(siRNA),实验组加入100 nmol·L^-1针对人VEGFR2的siRNA。用liposome转染阴性对照组和实验组的siRNA至原代HUVECs中,以流式细胞术测定细胞的纯度;以实时荧光定量聚合酶链式反应检测siRNA的基因沉默效率;划痕法检测细胞迁移距离;小管形成法检测细胞血管生成。结果原代HUVECs纯度高达(99.96±1.28)%。空白组、阴性对照组和实验组的基因沉默效率分别为(0.00±3.25)%,(5.56±2.32)%和(82.41±5.29)%;空白组、阴性对照组和实验组的迁移愈合率分别为(92.72±2.54)%,(85.67±4.32)%和(28.21±4.57)%;空白组、阴性对照组和实验组的总小管分支长度分别为(4639.87±600.35),(4441.92±584.82)和(791.59±106.29)μm。上述指标:实验组与空白组和阴性对照组相比,差异均有统计学意义(均P<0.01)。结论RNA干扰VEGFR2可抑制原代人脐静脉内皮细胞迁移及血管生成。

Objective To study the effect of RNA interfering vascular endothelial cell growth factor receptor 2(VEGFR2)on the migration and angiogenesis of primary human umbilical vein endothelial cells(HUVECs).Methods Primary HUVECs were extracted from neonatal umbilical cord.The cells were divided into three groups:blank group,negative control group and experimental group.Cells in the blank group were not treated;the negative control group is the no homologous small interfering RNA(siRNA)for human and mouse genes with a concentration of 100 nmol·L^-1;while the experimental group is the siRNA for human VEGFR2 with a concentration of 100 nmol·L^-1.Liposome transfected siRNA from the negative control group and the experimental group into the primary HUVECs.Purity determination of cells by flow cytometr.The silencing efficiency of siRNA was detected by real-time quantitative polymerase chain reaction.Cell migration distance was detected by scratch method.Cell angiogenesis was detected by tube formation assay.Results The purity of HUVECs was up to(99.96±1.28)%.Gene silencing efficiency in blank group,negative control group and experimental group were(0±3.25)%,(5.56±2.32)%,(82.41±5.29)%,respectively;the scratch healing rate in blank group,negative control group and experimental group were(92.72±2.54)%,(85.67±4.32)%,(28.21±4.57)%,respectively;the total length of tubular branches in blank group,negative control group and experimental group were(4639.87±600.35),(4441.92±584.82),(791.59±106.29)μm.Comparison between experimental group and blank group,negative control group,the difference of the factors were significantly(all P<0.01).Conclusion RNA interfered VEGFR2 gene can inhibit primary HUVECs migration and angiogenesis.