携人血栓调节蛋白基因内皮祖细胞预防动脉成形术后再狭窄

Local transplantation of human thrombomodulin gene - modified endothelial progenitor cells to pre- vent restenosis after angioplasty

目的探讨携人血栓调节蛋白（hTM）基因的内皮祖细胞（EPCs）局部移植预防动脉成形术后再狭窄的可行性。方法利用密度梯度离心法分离兔外周血EPCs；慢病毒载体plenti6.3-hTM-IRES-增强型绿色荧光蛋白（EGFP）感染EPCs，并以Fe2O3磁性纳米颗粒标记；双腔Fogarty球囊制作兔右侧颈总动脉损伤模型共36只，随机平均分为3组，每组分别移植hTM-EPCs、单纯EPCs及等量生理盐水至损伤血管段；术后1周每组随机选取4只兔取损伤血管段行Western blot检测及普鲁士蓝染色，观察血管损伤处有无外源性EPCs定植；术后4、8周行磁共振（MR）扫描，评估血管腔狭窄程度；术后8周行苏木素-伊红（HE）、弹力纤维及增殖细胞核抗原（PCNA）染色，观察损伤段血管壁结构、平滑肌及内膜增生程度。结果普鲁士蓝染色结果显示含铁EPCs附着于损伤处血管内膜；Western blot检测结果示hTM-EPCs组损伤血管段高表达hTM；MR结果显示hTM-EPCs组损伤血管段管腔狭窄程度低于单纯EPCs组及对照组；HE、弹力纤维及PCNA染色结果显示：术后8周，hTM-EPCs组、单纯EPCs组及对照组内膜/中膜比（N/M）分别为0.348±0.040、0.704±0.060和1.294±0.051，组间差异有统计学意义（P=0.000）；管腔狭窄率分别为（11.4±2.3）%、（24.3±4.8）%、（35.2±9.4）%（P=0.005）；PCNA阳性细胞指数分别为（12.3±3.9）%、（23.4±4.2）%、（49.3±6.8）%（P=0.000）。结论双腔Fogarty球囊阻断局部血流后，定向移植携hTM基因的EPCs可有效抑制血管成形术后再狭窄。

ObjectiveTo investigate whether restenosis can be inhibited by human thrombomodulin （hTM） gene-modified endothelial progenitor cells （EPCs） after angioplasty.MethodsEPCs derived from rabbit periphery blood were modified with hTM gene using lentiviral vector and were incubated with Fe2O3-arginine. Thirty-six New Zealand rabbits were randomly divided into 3 groups. Angioplastywas underwent by Fogarty ballooncatheter via catheterization of right common carotid artery, and then hTM-EPCs, EPCs or equal volume of normal saline were transplanted to the injury sites with blood flow blocked respectively. Histology and Western blotting were performed on 1th week for 4 rabbits from each group to evaluate cell adherence. Magnetic resonance （MR） was performed on 4th and 8th week after transplantation to evaluate restenosis. All of the rest rabbits were sacrificed at the end of 8th week after injury and the injured vessels were harvested for hematoxylin and eosin （HE）, elastic tissue and proliferating cell nuclear antigen （PCNA） staining to observe the vascular wall structure and degree of intimal hyperplasia and smooth muscle proliferation.ResultsPrussian blue staining showed iron-labeled cells distributed in the injured artery intima. Western blotting indicated that injured vascular transplanted with hTM-EPCs overexpressed thrombomodulin than other two groups. MR showed that the stenotic degree of the lumen in hTM-EPCs group was less serious than other two groups. The histopathological study on 8th week showed that the neointimal/media ratio （N/M） in hTM-EPCs group, EPCs group and control group was 0.348±0.040, 0.704±0.060 and 1.294±0.051, respectively （P=0.000）; the ratio of restenosis in each group was （11.4±2.3）%, （24.3±4.8）% and （35.2±9.4）%, respectively （P=0.005）; and the PCNA positive cell index in each group was （12.3±3.9）%, （23.4±4.2）% and （49.3±6.8）%, respectively （P=0.000）.ConclusionThe local transplantation of hTM-EPCs can prevent restenosis through Fogarty balloon after angioplasty in rabbit model.