RNA干扰抑制DLL4基因表达对人血管内皮细胞增殖的影响

Effects of RNA interference to Delta-like 4 expression on proliferation of human vascular endothelial cells

背景:Delta-like4(DLL4)是近年新发现的血管生长调控因子,研究表明抑制DLL4可导致过度无效的血管生成。目的:研究RNA干扰抑制DLL4基因表达对人血管内皮细胞增殖的影响。设计、时间及地点:随机分组,对比观察,于2007-07/2008-09在南昌大学医学院和江西省医学生物高技术重点实验室完成。材料:人微血管内皮细胞由上海细胞生物和生物化学研究所提供。方法:设计和化学合成靶向DLL4基因的siRNA序列,lepofectamineTM2000介导DLL4siRNA转染人微血管内皮细胞。提取细胞总RNA,进行反转录-聚合酶链反应扩增,琼脂糖电泳检测DLL4mRNA。提取细胞总蛋白,进行聚丙烯酰胺凝胶电泳。采用四甲基偶氮唑盐法检测DLL4siRNA对人微血管内皮细胞增殖的影响,实验分4组:①85nmol/LDLL4siRNA-duplex组。②85nmol/LDLL4siRNA-scrambled阴性对照组。③lepofectamine组。④未经处理组。主要观察指标:RNA干扰后,人微血管内皮细胞DLL4的转录和蛋白表达及人微血管内皮细胞的增殖。结果:反转录-聚合酶链反应和Westernblot实验结果显示,人微血管内皮细胞转录DLL4和表达DLL4蛋白;85nmol/LDLL4siRNA完全抑制人微血管内皮细胞DLL4转录和DLL4蛋白的表达。DLL4siRNA抑制人微血管内皮细胞DLL4表达的有效浓度为85nmol/L。四甲基偶氮唑盐实验结果显示,85nmol/L DLL4 siRNA-duplex组的细胞增殖率为190.00%,未经处理组的细胞增殖率为110.00%。结论:人微血管内皮细胞表达DLL4,DLL4siRNA抑制人微血管内皮细胞表达DLL4并促进人微血管内皮细胞增殖,DLL4对人微血管内皮细胞增殖具有抑制作用。

BACKGROUND： Delta-like 4 （DLL4） is a newly found regulating factor in angiogenesis. Studies demonstrated that inhibiting DLL4 can result in ineffective vascularization. OBJECTIVE： To explore the effects of RNA interference to DLL4 expression on proliferation of human vascular endothelial cells. DESIGN, TIME AND SETTING： A randomized controlled group of contrast observation was performed at the Medical College of Nanchang University and Key Laboratory of Medical Biology of Jiangxi Province from July 2007 to September 2008. MATERIALS： The human microvascular endothelial cells （HMECs） were supplied by Institute of Biochemistry and Cell Biology, Shanghai Institute for Biological Sciences, Chinese Academy of Sciences. METHODS： The siRNAs targeting to DLL4 gene were designed and chemically synthesized. DLL4 siRNAs were transfected into HMEC by lepofectamineTM2000. Total RNA was extracted from HMECs. Following RT-PCR, agar gel electrophoresis was performed to detect DLL4 mRNA. Total cellular protein was extracted and analyzed by polyacrylarnide gel electrophoresis. MTT was used to observe the effect of DLL4 siRNA on HMECs proliferation. Cells were divided into 85 nmol/L DLL4 siRNA-duplex group, 85 nmol/L DLL4 siRNA -scrambled negative group, lepofectamine group and no treatment group. MAIN OUTCOME MEASURES： The transcription and expression of DLL4, as well as proliferation of HMECs proliferation following RNA interference. RESULTS： The results of RT-PCR and Western blot indicated that DLL4 was expressed in HMECs at levels of mRNA and protein, which completely inhibited by 85 nmol/L of DLL4 siRNA. The effective concentration of DLL4 siRNA for HMECs was 85 nmol/L. MTT results showed that the ratio of proliferation was 190.00% in the 85 nmol/L DLL4 siRNA-duplex group, which was 110.00% in the no treatment group. CONCLUSION： DLL4 can be expressed in HMECs. DLL4 siRNAs inhibit the expression of DLL4 in HMECs, meantime promote HMECs proliferation. DLL4 has inhibitive effect on HMECs proliferation.