选择性清除单核/巨噬细胞对血管损伤后内膜增生影响的研究

Effects of macrophage depletion on neointimal formation after vascular injury

目的应用脂质体输送系统选择性清除单核/巨噬细胞,探讨血管损伤后抑制内膜增生的有效途径。方法采用反相蒸发技术将clodronate用脂质体包裹,在36只大鼠颈动脉损伤模型前1 d、损伤后6 d,分别尾静脉注射15 mg/kg体重的脂质体包裹clodronate(LC组,12只)、clodronate溶液(FC组,8只)、等量生理盐水(saline组,9只)或等量空脂质体(EL组,7只),14天后颈总动脉切片行HE、VG染色,用图象分析软件测定颈动脉管腔面积、增生内膜面积、中膜面积,并计算增生内膜与中膜面积比值(N/M)、管腔狭窄率(%)。另取10只大鼠尾静脉注射荧光标记的脂质体包裹clodr- onate(5只)或等量荧光标记的空脂质体(5只),颈总动脉损伤后1天下腔静脉抽血分离白细胞,并取颈总动脉、肝脏、脾脏,冰冻切片后在激光扫描共聚焦显微镜下观察荧光物质分布。结果包裹、未包裹clodronate的脂质体、罗丹明标记脂质体的平均直径分别为(213±20)nm、((19.8±14)nm、(209±19)nm,包裹率17.6%～19.0%,脂质体中clodronate浓度(7.6±1.1)mg/ml;各组大鼠损伤动脉内膜均有明显增生,但LC组血管内膜增生明显较其它3组明显减轻,表现为N/M、管腔狭窄率(%)明显低于其它3组相应值(P<0.01);FC组或EL组对血管内膜增生无明显影响(与saline组比,P>0.05)。各组间颈总动脉中膜面积无明显差别。尾静脉注射荧光标记脂质体48 h后,大鼠左颈总动脉、静脉血中白细胞、脾脏、肝脏中可以发现单核/巨噬细胞中有荧光标记物积聚,其中LC组大鼠上述组织中荧光标记物较EL组者明显减少,表明脂质体包裹clodronate可明显减少体内单核/巨噬细胞数量。结论通过clodronate的脂质体输送系统将巨噬细胞暂时灭活、清除,可以预防血管损伤后的血管再狭窄。

Objective The present study examined the role of monocytes/macrophages in vascular repair by means of systemic administration of clodronate in a liposomal delivery system. Methods Clodronate was encapsulated with liposomes using the reverse-phase evaporation technique. The carotid injury model was performed in 36 rats. Liposome clodronate was administered on days -1 and ＋ 6 intravenously via the tail vein. Free clodronate, saline, and empty liposomes were injected to control animals. Animals were euthanized by ether at 14 days after injury. After Verhoeff＇s elastin stain of artery sections, the residual lumen, the area bounded by the internal elastic lamina, and the area circumscribed by the external elastic lamina were measured directly. Neointimal thickening was expressed as the ratio between the neointimal to medial area （N/M）, and the ratio between the area of the neointimal and the original lumen （% stenosis）. A separate group of 10 rats was used for this study. Rats were injected with rhodamine-labeled liposome clodronate or buffer at day -1. Carotid arteries, bloods, livers and spleens were harvested and observed by confocal microscopy at day 1 after carotid artery injury. Results the study showed that the prepared liposomes had a high encapsulation efficiency of clodronate （ 17.6-19.0% ） with an average size of 200 nm. Marked neointimal formation and decreased luminal area were observed in all groups of animals. Liposome clodronate suppressed intimal growth when administered i.v. on days -1 and ＋ 6. Both N/M ratios and % stenosis in liposome clodronate group reduced significantly. No significant differences were found between treatment by empty liposomes, saline, or free clodronate in solution. Confocal microscopy images depicting disposition of fluorescent liposomes in arteries and in blood monocytes, liver, and spleen of rats 24 hours after injury. Reduction of monocytes and macrophages was detected by injection of fluorescent liposomes. Marked reduction of the fluorescent signal was observed in the injured arterial wall, in blood monocytes, in the liver and spleen of liposome clodronate treated animals. Conclusions Modulation of systemic inflammatory response after vascular injury affects the local inflammatory process and may prove to a valuable target in reducing excessive repair that lead to restenosis.