Effect Of Human Tissue Factor Pathway Inhibitor Gene Delivery In Vein Grafts On Thrombosis And Neointima Formation In Bypass Grafting Models

ExperimentⅠConstruction and identification of eukaryotic expressive plasmid vector pCMV-(Kozak)TFPIObjective To construct and identify eukaryotic expressive plasmid vector pCMV-(Kozak)TFPI aims to further study on antirestenosis effect of TFPI gene transferring into endotheliocytes in vein vessels. Methods The full length human tissue factor pathway inhibitor (TFPI) gene was extracted by RT-PCR. The Kozak sequence and subclone sites were led in TFPI gene. Subsequently, the (Kozak)TFPI sequence was connected with T vector for sequencing. After sequencing, the (Kozak)TFPI fragment was inserted in eukaryotic expressive plasmid vector pCMV. Moreover, the correctness of this pCMV- (Kozak)TFPI was verified by digestion of restriction enzymes. Results Sequencing result showed that the sequence of full length human TFPI gene was perfectly coincident with that in GeneBank, and Kozak sequence was led in. The digestion of restriction enzymes identified that the (Kozak)TFPI sequence was cloned into eukaryotic expressive plasmid vector pCMV. Conclusion The eukaryotic expressive plasmid vector pCMV-(Kozak)TFPI was constructed successfully. ExperimentⅡDetection of exogenous tissue factor pathway inhibitor gene mRNA and protein expressed in human umbilical vein endotheliocytesObjective To evaluate the expressive ability of pCMV-(Kozak)TFPI vector, we detect the expressive effect of eukaryotic expressive plasmid vector pCMV-(Kozak)TFPI in human umbilical vein endotheliocytes (HUVECs). Methods Twenty-four wells in four 6-well culture plates in which HUVECs were growing were medially divided into 2 groups with 12 holes in each group: TFPI group and empty plasmid group. HUVECs in TFPI group were transfected with cationic liposome containing the vector pCMV-(Kozak)TFPI. In empty plasmid group, vector pCMV-(Kozak)TFPI was replaced by empty plasmid pCMV, and other conditions were all identical. Twenty-four hours later, RT-PCR, immunofluorescence and Western blot were applied to detect expression of exogenous TFPI mRNA and protein in HUVECs. The transfection rate of exogenous TFPI gene was calculated by immunofluorescence results. Results RT-PCR, immunofluorescence and Western blot detected expression of exogenous TFPI mRNA and protein in HUVECs in TFPI group. The transfection rate of exogenous TFPI gene is 23%. Conclusion The exogenous TFPI mRNA and protein were expressed successfully by eukaryotic expressive plasmid vector pCMV-(Kozak)TFPI in transfected HUVECs. ExperimentⅢEffect of human tissue factor pathway inhibitor gene transfection in vein grafts on thrombus formation and early patency rate in rabbitsObjective To reduce thrombus formation after coronary artery bypass grafting, we investigate the antithrombotic effect of human TFPI gene delivery on vein grafts in rabbits. Methods Sixty Japanese White rabbits were randomly divided into 3 groups with 20 rabbits in each group: TFPI group, empty plasmid control group and empty control group. Animal model of common carotid artery bypass grafting was constructed. Before anastomosis, vein endotheliocytes in TFPI group were transfected with cationic liposome containing the plasmid pCMV-(Kozak)TFPI (400μg) by pressurizing infusion (30min). In empty plasmid control group, vector pCMV-(Kozak)TFPI was replaced by empty plasmid pCMV (400μg). While, those endotheliocytes in empty control group weren't interfered. After operation, vein grafts were harvested at third day for RT-PCR, Western blot and immunohistochemical analyses of exogenous gene expression and for pathological, scanning electron microscope observation of thrombus. At 30th day, the patency rate was recorded by vessel Doppler ultrasonography. Results Three days after operation, human TFPI mRNA and protein were detected in TFPI gene transferred vein grafts. Thrombosis was found in 8 animals of empty plasmid control group and in 7 animals of empty control group, but in only 1 of the TFPI group (P<0.05). Thirty days after operation, 5 vein grafts were occluded in both empty plasmid control group and empty control group, but none of vein grafts were occluded in TFPI group (P<0.05). Three days after operation, the endothelial surfaces of the vein grafts in both control groups were covered with aggregated erythrocytes and platelets, and it couldn't be seen in TFPI group. Conclusion Human tissue factor pathway inhibitor gene transfection reduces thrombus formation and improves early patency rate in vein grafts in rabbits. ExperimentⅣInfluence of human tissue factor pathway inhibitor gene transfection in vein grafts on neointima formation in rabbitsObjective To reduce restenosis in vein grafts after coronary artery bypass grafting, we investigate the effect of TFPI gene delivery on neointima formation in rabbits. Methods Sixty Japanese White rabbits were randomly divided into 3 groups with 20 rabbits in each group: TFPI group, empty plasmid control group and empty control group. Animal model of common carotid artery bypass grafting was constructed. Before anastomosis, vein endotheliocytes in TFPI group were transfected with cationic liposome containing the plasmid pCMV-(Kozak)TFPI (400μg) by pressurizing infusion (30min). In empty plasmid control group, vector pCMV-(Kozak)TFPI was replaced by empty plasmid pCMV (400μg). While, those endotheliocytes in empty control group weren't interfered. After operation, vein grafts were harvested at third day for RT-PCR, Western blot and immunohistochemical analyses of exogenous gene expression and at 30th day for histopathology measurement of intimal areas, media areas and calculation of intimal/media area ratios. Luminal diameter and vessel wall thickness were also measured by vessel Doppler ultrasonography and cellular category of neointima was analyzed by transmission electron microscope at 30th day after operation. Results Three days after operation, human TFPI mRNA and protein were detected in TFPI gene transferred vein grafts. Thirty days after operation, the mean luminal diameter of the TFPI group(2.68±0.32mm)was greater than those of the empty plasmid control group(2.41±0.23mm) and empty control group(2.38±0.21mm)(P<0.05), and vessel wall thickness of TFPI group(1.09±0.11mm)was markedly smaller than those of the two control groups(1.28±0.16mm and 1.34±0.14mm)(P<0.01). The mean intimal area, the ratio of the intimal/media areas in the TFPI group(0.62±0.05mm2, 0.51±0.08)were reduced compared with those of the two control groups(0.70±0.05mm2, 0.72±0.04mm2, P<0.05 and 0.58±0.06, 0.59±0.08, P<0.05), but mean media areas had no significant differences among three groups (1.23±0.11mm2, 1.22±0.11mm2 and 1.23±0.12mm2, P>0.05). Through transmission electron microscope analyses, no smooth muscle cells were seen in neointima of TFPI group in many visual fields, but smooth muscle cells were found in neointima of two control groups. Conclusion Human tissue factor pathway inhibitor gene transfection reduced intimal thickness in vein grafts in rabbits.