利用RNAi技术下调VEGF表达对血管瘤内皮细胞影响.doc

利用RNAi技术下调VEGF表达对血管瘤内皮细胞影响[摘要]目的：培养人血管瘤内皮细胞（HemECs），利用RNA干扰技术下调HemECs 中VEGF的表达，观察其对血管瘤内皮细胞的影响。 方法：利用组织块法培养HemECs，鉴定、筛选和纯化后，分为A、B、C三组，分别为转染组、空脂质体组和对照组。利用脂质体法将pGCsiU6/Neo/dsRED-VEGF真核表达质粒转染到内皮细胞中。通过荧光显微镜大体观察转染效率和细胞生长情况、ELISA法检测细胞分泌VEGF蛋白和MTT法检测质粒转染对HemECs的影响。 结果：转染组第五天转染细胞达到总细胞80%以上，但是细胞凋亡逐渐增加。第七天细胞稀少，漂浮死细胞增多。转染阳性细胞减少，细胞整体状态差。转染组培养上清中VEGF蛋白浓度在转染的第1、3、5、7天分别为141.3±7.82、92.34±5.71、68.07±5.95及40.65±6.71pg/ml。MTT法检测各组490nm的OD值在1、3、5、7天均有统计学差异（P＜0.05）。 结论：转染后的细胞活性降低，通过对其分泌的VEGF蛋白的测量发现，转染组VEGF蛋白明显下降，同时，细胞活力受到影响，漂浮的死细胞增多。这些都可以说明，VEGF在血管瘤内皮细胞生长、增殖之中起着非常重要的作用，这也为我们治疗血管瘤提供一个新的靶点。 [关键词]RNAi，血管瘤内皮细胞，VEGF，细胞转染 [中图分类号]R732.2 [文献标识码]A [文章编号]1008-6455（2011）01-0089-04 Effect of down-regulated VEGF by RNAi on the HemECs MA Ge-jia,YI Cheng-gang,LIU Bei,YANG Li (Institute of Plastic Surgery,Xijing Hospital,The Fourth Military Medical University,Xi’an 710032,Shaanxi,China) Abstract:ObjectiveDown regulating the VEGF by RNAi in the cultured HemECs to detect its effect on HemECs.MethodsCulturing HemECs though the tissue explants adherant method,after identification,screening,and purification,dividing the HemECs into A,the transfection group,B,the mock,and C the control group,using liposome to transfect pGCsiU6/Neo/dsRED-VEGF into epithelial cells,and analysis the efficacy of transfection and thecell proliferation by fluorescent microscopy,testing the VEGF secretion level by ELISA and HemECs proliferation by MTT after transfection.Results The tranfected cells in group A occupied over 80% overall cell on the fifth day,at the same time the apoptosis of cells increased,and on the seventh day these was a few cells left,the fluorescent positive cells decreased, the cell overall situation was weak. The VEGF level in the culture supernatant by ELISA was 141.3±7.82、92.34±5.71、68.07±5.95 and 40.65±6.71pg/ml after the transfection day 1、3、5、7 respectively. MTT assay showed that the OD value after the transfection the day 1,3,5,7 was statistically significant (P＜0.05).ConclusionThe cel