人组织因子途径抑制因子基因转染对移植静脉新生内膜的影响

INFLUENCE OF HUMAN TISSUE FACTOR PATHWAY INHIBITOR GENE TRANSFECTION ON NEOINTIMA FORMATION IN VEIN GRAFTS

目的为减少冠状动脉旁路移植术后移植静脉再狭窄,探讨人组织因子途径抑制因子(tissuefactorpathwayinhibitor,TFPI)基因转染对移植静脉内膜增生的影响。方法构建真核表达质粒pCMV-(Kozak)TFPI。将48只日本大耳白兔随机分为3组,每组16只,即TFPI转染组、空载体对照组和空白对照组。建立颈总动脉旁路移植模型。吻合前,TFPI转染组移植静脉内采用阳离子脂质体pCMV-(Kozak)TFPI(400μg)和腔内加压灌注法(30min)转染,空载体对照组以空质粒pCMV(400μg)代替pCMV-(Kozak)TFPI,空白对照组不予干预。术后3d,用RT-PCR、Western-blot和免疫组织化学法检测外源基因在移植静脉中的表达。术后30d,血管多普勒测量移植静脉管腔内径和管壁厚度;组织病理标本测量内膜面积和中膜面积,并计算其比值;透射电镜观察移植静脉新生内膜的细胞构成。结果TFPI转染组移植静脉中有人TFPI基因mRNA和蛋白表达,两对照组未见表达。TFPI转染组移植静脉管腔内径为(2.68±0.32) mm,大于空载体对照组(2.41±0.23)mm和空白对照组(2.38±0.21)mm,差异均有统计学意义(P<0.05)。TFPI转染组管壁厚度为(1.09±0.11) mm,小于空载体对照组(1.28±0.16)mm和空白对照组(1.34±0.14)mm,差异均有统计学意义(P<0.01)。TFPI转染组移植静脉内膜面积和内膜、中膜面积比值分别为(0.62±0.05)mm2及0.51±0.08,均小于两对照组的(0.70±0.05) mm2、0.58±0.06及(0.72±0.04) mm2、0.59±0.08(P<0.05);中膜面积各组差异无统计学意义(P>0.05)。透射电镜观察,TFPI转染组移植静脉内膜未见平滑肌细胞,两对照组均见平滑肌细胞。结论人TFPI基因转染减少移植静脉内膜增生。

Objective To reduce restenosis in vein grafts after coronary artery bypass grafting, to investigate the effect of human tissue factor pathway inhibitor（TFPI） gene delivery on neointima formation. Methods The eukaryotic expressed plasmid vector pCMV-（Kozak） TFPI was constructed. Forty-eight Japanese white rabbits were randomly divided into 3 groups with 16 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 were transfected with cationic liposome containing the plasmid pCMV- （Kozak） TFPI （400 μg） by pressurizing infusion （30 rain） in TFPI group. In empty plasmid control group, vector pCMV- （Kozak） TFPI was replaced by empty plasmid pCMV （400 μg）. In empty control group, those endotheliocytes were not interfered. After operation, vein grafts were harvested at 3 days for immunohistochemical, RT- PCR and Western-blot analyses of exogenous gene expression and at 30 days for histopathology measurement of intimal areas, media areas and calculation of intimal/media areas ratio. 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 30 days after operation. Results Human TFPI mRNA and protein were detected in TFPI group. The mean luminal diameter of the TFPI group, empty plasmid control group and empty control group was （2.68 ± 0.32） ram, （2.41 ± 0.23） mm and （2.38 ± 0.21） mm respectively. There were statistically significant differences between TFPI group and control groups （P 〈 0.05）. The vessel wall thickness of the TFPI group, empty plasmid control group and empty control group was （1.09 ± 0.11） mm, （1.28 ± 0.16） mm and （1.34 ± 0.14） mm respectively. There were statistically significant differences between TFPI group and other Control groups （P 〈 0.01）. The mean intimal areas, the ratio of the intimal/media areas of the TFPI group were （0.62 ± 0.05） mm^2and 0.51 ± 0.08 respectively,whichwere reduced compared withthose of the two control groups（P 〈 0.05）. The mean media areas had no significant differences among three groups （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 TFPI gene transfection reduced intimal thickness in vein grafts.