Transplantation of VEGF165-gene-transfected endothelial progenitor cells in the treatment of chronic venous thrombosis in rats  

作　　者：MENG Qing-you LI Xiao-qiang YU Xiao-bin LEI Feng-rui JIANG Kun LI Chuan-yong 

机构地区：[1]Department of Vascular Surgery, Second Affiliated Hospital of Suzhou University, Suzhou, Jiangsu 215004, China

出　　处：《Chinese Medical Journal》2010年第4期471-477,共7页中华医学杂志（英文版）

基　　金：This work was supported by a grant from the Natural Science Foundation of Jiangsu Province （No. BK2007055）.

摘　　要：Background The organization and recanalization of thrombi is a dynamic and complex process. The aim of this research was to study the cotherapeutic effect of stem cell transplantation and gene transfection on chronic venous thrombosis. Methods We constructed a recombinant adenoviral vector carrying the vascular endothelial growth factor 165 （VEGF165） gene by using the pAdEasy system, which was subsequently identified and amplified. Simultaneously, endothelial progenitor cells （EPCs） were isolated from rat bone marrow using Ficoll, cultured in EBM-2MV medium, and identified. Then, the cells were transfected with the recombinant Ad-VEGF165. The EPCs were labeled with 1 ,1＇-dioctadecyl-3,3,3＇,3＇-tetramethylindocarbocyanine （Dil） before transplantation. A rat model of chronic vein thrombosis was developed by partial ligation of the inferior vena cava. The rats were randomly divided into 4 groups （n=25, each）： A, Ad-VEGF165/EPC-transplantation group received 1 ml （10^6） of Ad-VEGF165/EPCs; B, EPC-transplantation group received 1 ml （10^6） of EPCs; C, Ad/EPC-transplantation group received 1 ml （10^6） of Ad/EPCs; D, control group received 1 ml of the transplantation medium. The thrombi and adjacent caval walls were harvested 28 days after transplantation; real-time quantitative polymerase chain reaction was used to detect the expression level of vascular endothelial growth factor （VEGF） mRNA; and western blotting was used to measure changes in VEGF protein expression. Hematoxylin-eosin staining and immunohistochemical staining were performed to detect recanalization. Neovascularization was detected by immunohistochemical staining using the antibody for von Willebrand factor （vWF）, which is a component of endothelial cells.The capillary density was quantitatively determined by counting the capillaries under a high-power microscope. Results The Ad-VEGF165 was constructed, and bone-marrow-derived EPCs were cultivated and successfully identified. We determined the optimum transfecBackground The organization and recanalization of thrombi is a dynamic and complex process. The aim of this research was to study the cotherapeutic effect of stem cell transplantation and gene transfection on chronic venous thrombosis. Methods We constructed a recombinant adenoviral vector carrying the vascular endothelial growth factor 165 （VEGF165） gene by using the pAdEasy system, which was subsequently identified and amplified. Simultaneously, endothelial progenitor cells （EPCs） were isolated from rat bone marrow using Ficoll, cultured in EBM-2MV medium, and identified. Then, the cells were transfected with the recombinant Ad-VEGF165. The EPCs were labeled with 1 ,1＇-dioctadecyl-3,3,3＇,3＇-tetramethylindocarbocyanine （Dil） before transplantation. A rat model of chronic vein thrombosis was developed by partial ligation of the inferior vena cava. The rats were randomly divided into 4 groups （n=25, each）： A, Ad-VEGF165/EPC-transplantation group received 1 ml （10^6） of Ad-VEGF165/EPCs; B, EPC-transplantation group received 1 ml （10^6） of EPCs; C, Ad/EPC-transplantation group received 1 ml （10^6） of Ad/EPCs; D, control group received 1 ml of the transplantation medium. The thrombi and adjacent caval walls were harvested 28 days after transplantation; real-time quantitative polymerase chain reaction was used to detect the expression level of vascular endothelial growth factor （VEGF） mRNA; and western blotting was used to measure changes in VEGF protein expression. Hematoxylin-eosin staining and immunohistochemical staining were performed to detect recanalization. Neovascularization was detected by immunohistochemical staining using the antibody for von Willebrand factor （vWF）, which is a component of endothelial cells.The capillary density was quantitatively determined by counting the capillaries under a high-power microscope. Results The Ad-VEGF165 was constructed, and bone-marrow-derived EPCs were cultivated and successfully identified. We determined the optimum transfec

关 键 词：vascular endothelial growth factor endothelialprogenitor cell gene transfection ADENOVIRUS venous thrombosis 

分 类 号：Q78[生物学—分子生物学] S78[农业科学—林学]