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Effect of Human Tissue Factor Pathway Inhibitor Gene Delivery in Vein Grafts on Thrombosis and Neointima Formation in Bypass Grafting Models

Author: LiQuan
Tutor: ZhangKaiLun
School: Huazhong University of Science and Technology
Course: Surgery
Keywords: Coronary artery bypass grafting Tissue factor pathway inhibitor Gene Carrier Transfection Endothelial cells Vein graft Thrombus Restenosis
CLC: R543
Type: PhD thesis
Year: 2008
Downloads: 45
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Abstract

Experiment eukaryotic expression vector pCMV-(Kozak) TFPI Construction and identified the purpose of building and identification of eukaryotic expression vector pCMV-(Kozak) of TFPI, anti-restenosis studies lay the foundation for the next transfected vein endothelial cells. Methods of RT-PCR method to extract the full-length human tissue factor pathway inhibitor (TFPI) gene, the introduction of a Kozak sequence, and subcloning sites (Kozak) TFPI sequence connected into the T vector and sequenced. (Kozak) TFPI sequence and then subcloned into pCMV plasmid, restriction enzyme digestion. The results of the sequencing of the full-length human TFPI gene sequences in GeneBank sequences were identical Kozak sequence successfully joined. Enzyme digestion (Kozak) TFPI sequence successfully cloned into pCMV. Conclusion We successfully constructed pCMV-(Kozak) TFPI eukaryotic expression vector. Second experiment exogenous TFPI gene mRNA and protein detection purpose expression in human umbilical vein endothelial cells to detect eukaryotic expression vector pCMV-(Kozak) TFPI expression in human umbilical vein endothelial cells (HUVEC), to evaluate the carrier The ability to express. Method long HUVEC four 6-well plates, 24-hole average divided into two groups, namely TFPI transfection group and empty vector transfected group, 12 in each hole. TFPI group in cationic liposome-mediated transfection, pCMV-(Kozak) TFPI transfected HUVEC, empty vector transfected group by empty vector pCMV instead pCMV-(Kozak) TFPI transfected HUVEC, and the remaining conditions are the same . 24h after transfection, HUVEC exogenous TFPI mRNA and protein expression was detected by RT-PCR, immunofluorescence and Western blot and immunofluorescence results exogenous TFPI gene transfection efficiency. RT-PCR, immunofluorescence and Western blot to TFPI transfection group of exogenous TFPI mRNA and protein expression. Exogenous TFPI gene transfection rate was 23%. Conclusion eukaryotic expression vector pCMV-(Kozak) TFPI in HUVEC transfected successful expression of the exogenous TFPI mRNA and protein. The experimental trio TFPI gene transfection to reduce coronary artery bypass graft thrombosis formation purpose and the impact of the recent patency rates of vein graft thrombosis formation explore human TFPI gene transfection to vein graft thrombosis generated. 60 Japanese white rabbits were randomly divided into three groups of 20 each, that TFPI transfection group, empty vector control group and blank control group. Carotid artery bypass grafting model, consistent, TFPI transfected group vein graft endothelial cells using cationic liposomes pCMV-(Kozak) TFPI (400μg) and cavity pressure perfusion (30min) transfected with empty vector control group with empty plasmid pCMV (400μg) instead of pCMV-(Kozak) TFPI, has not interfered with the blank control group vein endothelial. Postoperative 3d, using RT-PCR, Western blot and immunohistochemistry assay exogenous gene in the vein graft in expression, pathological specimens, scanning electron microscopy vein graft thrombosis. After surgery the 30d, vascular Doppler observed patency rates. Results Postoperative 3d, TFPI transfected allografts someone TFPI gene mRNA and protein expression in the vein. TFPI transfected group, an empty vector and blank control group, respectively, 8 and 7 vein graft thrombosis; postoperative 30d, above 0, 5 and 5 totally occluded vein graft, former intravenous thrombosis rate is lower than the latter two (P lt; 0.05), while the vein patency higher than the latter two (P lt; 0.05). Postoperative 3d, scanning electron microscopy showed that the two control groups endothelial surface of the red blood cell and platelet adhesion, aggregation, and TFPI transfection group is basically normal. The TFPI gene intervention Conclusions reduced rabbit transplant vein thrombosis, recent patency rates. Experiment four people TFPI gene transfection on the purpose of the impact of vein graft neointimal reduce coronary artery bypass grafting vein graft restenosis explore human TFPI gene transfection to the impact of the vein graft intimal hyperplasia. 60 Japanese white rabbits were randomly divided into three groups of 20 each, that TFPI transfection group, empty vector control group and blank control group. Carotid artery bypass grafting model, consistent, TFPI transfected group vein graft endothelial cells using cationic liposomes pCMV-(Kozak) TFPI (400μg) and cavity pressure perfusion (30min) transfected with empty vector control group with empty plasmid pCMV (400μg) instead of pCMV-(Kozak) TFPI, has not interfered with the blank control group vein endothelial. Postoperative 3d, RT-PCR, Western blot and immunohistochemistry assay exogenous gene expression in the vein graft. Postoperative 30d, vascular Doppler measurements lumen diameter and wall thickness of the vein graft. The histopathological specimens measuring intimal areas (I) and in the membrane area (M) and calculate (I / M). Transmission electron microscopy of cells in the vein graft neointimal. Results Postoperative 3d, TFPI transfected allografts someone TFPI gene mRNA and protein expression in the vein. Postoperative 30d TFPI transfection group transplanted venous lumen diameter (2.68 ± 0.32) mm, greater than the empty vector control group (2.41 ± 0.23) mm and blank control group (2.38 ± 0.21) mm (P lt; 0.05). The TFPI transfection Group wall thickness (1.09 ± 0.11) mm, less than the empty vector control group (1.28 ± 0.16) mm and blank control group (1.34 ± 0.14) mm (P lt; 0.01). TFPI vein graft I and I / M transfection group (0.62 ± 0.05) mm2 and 0.51 ± 0.08 respectively, were less than the control group (0.70 ± 0.05) mm2 mm2 (P lt; 0.05), (0.72 ± 0.04) and 0.58 ± 0.06,0.59 ± 0.08 (P lt; 0.05); M groups showed no significant difference (1.23 ± 0.11) mm2 (1.22 ± 0.11) mm2 and (1.23 ± 0.12) mm2 (P gt; 0.05). TEM observations TFPI-transfected group intravenous the intima multiple horizons is not found in the smooth muscle cells, smooth muscle cells more than the other two groups vision. Conclusion TFPI gene transfection to reduce vein graft intimal hyperplasia.

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CLC: > Medicine, health > Internal Medicine > Heart, blood vessels ( circulatory ) disease > Vascular disease
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