VEGF_(165)转染大鼠血管内皮细胞对新生血管形成影响的实验研究

Effect of VEGF_(165) transfected rat vascular endothelial cell on angiogenesis

目的探讨血管内皮细胞生长因子(vascular endothelial growth factor,VEGF)基因与增强绿色荧光蛋白(enhanced green fluorescent protein,EGFP)共表达载体转染大鼠血管内皮细胞,植入大鼠体内,观察诱导新生血管情况,为移植组织诱导新生血管形成奠定基础。方法对质粒pIRES2-EGFP/VEGF165进行扩增、纯化,以脂质体法转染大鼠血管内皮细胞并植入肾被膜下。采用荧光显微镜检测增强绿色荧光蛋白在内皮细胞中的表达,用流式细胞仪检测转染效率。用RT-PCR检测VEGF mRNA的表达,免疫组化检测VEGF在内皮细胞中的表达。切取移植肾脏组织标本,HE染色观察组织形态学变化。结果荧光显微镜观察到实验组内皮细胞有特异性的EGFP表达,流式细胞仪分析转染效率为13.06%。实验组血管内皮细胞胞核和胞浆中均有VEGF表达。RT-PCR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。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尽管血管内皮细胞仍存活,但未形成明显血窦。结论转染VEGF是促进内皮细胞早期(14d内)形成新生血管的有效途径。

Objective To study the effect of vascular endothelial growth factor（VEGF） transfected rat vascular endothelial cell on angiogenesis with enhanced green fluorescent protein（EGFP） carrier and transplanted rat in vivo, and to observe neovessels by its inducing for providing basis of transplanting tissue inducing neovessels. Methods The vascular endothelial cell was transfected by pIRES2-EGFP plasmid, and then transplanted it under the capsule of kidney. The EGFP expression was observed by fluorescence microscope. Flurocytometery were applied to evaluate the transfection efficiency. The VEGF mRNA expression was detected by RT-PCR. Immunohistochemical staining of human VEGF165 was used to determine the expression of VEGF in vessel endothelial cells. The histology and angiogenesis of kidney were studied with HE staining. Results There was specific EGFP expression in epithelial cells observed by fluromicroscope. The transfected ratio was 13.06 % determined by flurocytometery. In experimental group the VEGF expression was found in vascular cell nucleus and cytoplasm. The human orgin VEGF165 gene in vascular endothelial cells of experimental rat expressed at mRNA level was shown by RT-PCR. Fourteen days after transplantation,the neocapillary net angionenesis was found under the kidney capsule in experimental rot. Although the vascular endothelial cells were still alive, but markable blood sinus was not formed. Condusion The transfection of VEGF is an effective approach for promoting endothelial cells to form neovessels in early stage（within 14 days）.