腺病毒介导组织因子途径抑制物基因转染兔损伤颈动脉后的表达及对内膜增生的抑制作用

Ad-TFPI gene transfer attenuates intimal proliferation in rabbit carotid arteries after balloon injury

目的检测人组织因子途径抑制物（tissue factor pathway inhibitor，TFPI）基因腺病毒载体系统在体表达及对兔颈动脉球囊损伤后新生内膜增生的抑制作用。方法共采用中国大耳白兔70只。分为4组，分别为腺病毒介导TFPI基因（Ad-TFPI）转染组、腺病毒介导LacZ基因（Ad-LacZ）转染组、PBS组及假手术组，前3组分别于颈动脉球囊损伤后局部转染Ad-TFPI、Ad-LacZ或PBS，假手术组只分离颈总动脉，不做球囊损伤。Ad-TFPI组和Ad-LacZ组每组25只动物，PBS组及假手术组每组10只。Ad-TFPI组和Ad-LacZ组分别于术后3、7、10、14和28d各处死3只动物，用RT-PCR、ELISA方法，检测TFPI在颈动脉壁中的表达。术后4周时，4组剩余动物（每组10只）行颈动脉造影检测最小管腔直径，之后取血管标本，观察病理形态学变化，测算出血管新生内膜面积、内膜与中膜的比例（I／M）、管腔狭窄程度。结果基因转染后3d，Ad-TFPI组检测到TFPI mRNA及蛋白表达，10-14d为表达高峰，28d有所下降但仍可检测到，Ad-LacZ组未测到人TFPI的表达。基因转染后4周，颈动脉造影结果示Ad-TFPI组最小管腔直径明显大于PBS组和Ad-LacZ组[（1.17±0.43）mm比（0.43±0.33）mm、（0.39±0.24）mm]，管腔狭窄百分率明显低于PBS组和Ad-LacZ组[（32±8.2）％比（79±10.2）％、（81±13.1）％]，P均〈0.01。形态学检查分析结果显示Ad-TFPI组新生内膜面积明显小于PBS组和LacZ组[（0.17±0.03）mm^2比（0.73±0.03）mm^2、（0.78±0.04）mm^2]，I／M值明显小于PBS组和LaeZ组（1.02±0.25比2.76±0.33、2.92±0.24），管腔狭窄程度明显低于PBS组和LacZ组[（24.5±2.1）％比（78.2±2.8）％、（81.3±3.2）％]，P均〈0.01。结论人TFPI腺病毒表达载体可在体内有效表达，并且对血管成形术后再狭窄有抑制作用。

Objective To investigate the in vivo gene expression of adenovirus-mediated human tissue factor pathway inhibitor （hTFPI） and its inhibition effects on intimal proliferation in rabbit carotid arteries after ballon injury. Methods Rabbits underwent carotid artery balloon injuries were treated with Ad-TFPI （ n = 25 ）, Ad-LacZ （ n = 25 ） or PBS （ n = 10）, respectively. Sham operated rabbits （ n = 10 ） serve as normal controls. The expressions of human TFPI at mRNA and protein levels were detected by RT- PCR and ELISA respectively on the 3rd, 7th, 10th, 14th, 28th day after operation. Intimal proliferation was detected by angiograms and morphometric analysis. Results TFPI mRNA and protein expressions were detected at 3 days and peaked at the 10th and 14th day after TFPI gene transfer. The expressions were still detectable on the 28th day. There was no TFPI expression in Ad-LacZ group. The carotid angiogram results indicated that the minimal lumen diameter in TFPI group was significantly larger and the lumina stenosis percentage was significantly lower in TFPI group compared those in Ad-LacZ and PBS groups （ all P 〈 0. 05 ）. The morphometric analysis showed that the intimal area, the ratio of the intimal/media area, the lumina stenosis percentage in TFPI group were all significantly reduced compared with those in Ad-LacZ and PBS groups （all P 〈0.01）. Conclusions The TFPI gene could be effectively transferred by adenovirus vector to injured carotid arteries and transferred Ad-TFPI could significantly attenuate intimal proliferation in balloon injured carotid arteries in rabbits.