真核表达载体血管内皮生长因子转染血管内皮细胞及对新生血管形成的影响

Effect of vascular endothelial growth factor transfected vascular endothelial cells on angiogenesis

背景:血管内皮生长因子(vascular endothelial growth factor,VEGF)能与存在内皮细胞表面的特异性受体结合,刺激体内新生血管形成,但在体内半衰期极短,在移植局部难以维持足够的浓度,限制了其临床应用。目的:观察VEGF与增强绿色荧光蛋白(enhanced green fluorescent,EGFP)共表达载体转染对大鼠血管内皮细胞形成新生血管的影响。设计、时间及地点:随机分组设计、对照动物实验,于2004-09/2005-01在中国医科大学附属第一医院器官移植实验室完成。材料:30只雄性Wistar大鼠按随机数字表法分为3组,每组10只。质粒pIRES2-EGFP/VEGF165由第四军医大学提供。方法:采用层析法大量提取pIRES2-EGFP/VEGF165质粒,采用阳性脂质体法进行转染,计数1×106血管内皮细胞植入雄性Wistar大鼠肾被膜下。实验组:植入转染质粒pIRES2-EGFP/VEGF165的内皮细胞;空白转染组:植入转染质粒pIRES2-EGFP的内皮细胞;对照组:植入正常大鼠血管内皮细胞。主要观察指标:①转染72h后观察EGFP及VEGF表达情况。②流式细胞仪检测转染效率。③植入后14d苏木精-伊红染色观察植入血管内皮细胞处组织形态学变化。结果:30只大鼠均进入结果分析。①荧光显微镜下实验组内皮细胞有特异性的EGFP表达。②流式细胞仪分析转染效率为13.06%。③实验组血管内皮细胞胞核和胞浆中均有VEGF表达。反转录-聚合酶链反应显示实验组大鼠血管内皮细胞中有人源化VEGF165基因在mRNA水平表达。④移植后14d,实验组大鼠肾被膜下可见成团的新生毛细血管网形成,而对照组及空白转染组虽血管内皮细胞仍存活,但未形成明显血窦。结论:转染VEGF基因是促进内皮细胞早期(14d内)形成新生血管的有效途径。

BACKGROUND： Vascular endothelial growth factor （VEGF） combined with the specific receptor on surface of endothelial cell, may stimulate the angiogenesis in vivo, but it is difficult to maintain enough concentration in the transplant site due to the short half-life period, which thus limits the clinical application. OBJECTIVE： To investigate the effect of VEGF and enhanced green fluorescent （EGFP） co-expression vector transfected rat vascular endothelial cell （VEC） on the angiogenesis. DESIGN, TIME AND SETTING： A random grouping controlled animal experiment was carried out in the Laboratory of Organ Transplantation, the First Affiliated Hospital of China Medical University between September 2004 and January 2005. MATERIALS： Thirty male Wistar rats were randomly divided into three groups, and each group contained 10 animals. Plasmid pIRES2-EGFP/VEGF165 was offered by the Fourth Military Medical University of Chinese PLA. METHODS： The pIRES2-EGFP/VEGF165 plasmid was extracted with stratographic analysis method, and the VEC was transfected with liposome, then was transplanted into the capsule of rat kidney at the count of 1 ×10^6. VEC transplanted with pIRES2-EGFP/VEGF165 plasmid was taken as experiment group, while that transplanted with pIRES2-EGFP plasmid as blank transfection group and normal rat VEC as control group. MAIN OUTCOME MEASURES： The EGFP and VEGF expression was observed at 72 hours after transfection. Flow cytometry was used to calculate the transfection efficiency. Hematoxylin-eosin stain for kidney was used to evaluate the histology of VEC after 14 days of transplantation. RESULTS： Thirty rats were all involved in the result analysis. Under fluorescence microscope, specific EGFP expression was detected in the VEC of experiment group. The transfection efficiency was 13.06% determined by flow cytometry. The VEGF was expressed in both nucleus and endochylema of VEC in the experiment group. Reverse transcription-polymerase chain reaction showed VEGF165 mRNA expression in the VEC of the experiment group. At 14 days after transplantation, microvascular network formed as a cluster in the renal capsule of experimental rats. But no obvious blood sinus was seen in the control group and blank transfection group, though the transplanted VECs were still alive. CONCLUSION： The VEGF transfected rat VEC can effectively stimulate the angiogenesis in the early stage （14 days）.