包载雷帕霉素的聚乳酸-聚乙醇酸纳米粒子对小型猪冠状动脉损伤后平滑肌细胞增殖的影响及机制

Efficacy and mechanism of local delivery of rapamycin and rapamycin-loaded poly （lactic-coglycolic） acid nanoparticles on coronary restenosis of injury-stenosis model of minipigs

目的研究局部血管内灌注包载雷帕霉素（RPM）的聚乳酸-聚乙醇酸共聚物（PLGA）纳米粒子（NPs）对损伤-狭窄小型猪冠状动脉损伤后平滑肌增殖、基质金属蛋白酶（MMP）-2、基质金属蛋白酶抑制物（TIMP）-2及p27^kip1mRNA表达的影响。方法28头小型猪模型随机数字表法分为4组，每组7只，冠状动脉过大球囊损伤后经Dispatch^TM球囊局部分别灌注生理盐水、5．0mg／ml空白NPs、1．0mg／ml RPM和5．0mg／ml RPM—PLGA NPs。手术第30天复查造影并留取冠脉标本。评价干预前后各组冠脉造影血管狭窄程度，切片常规行苏木精-伊红、Weigert氏间苯二酚复红和苦味酸-天狼星红染色并行形态学比较。免疫组化染色法检测增殖细胞核抗原（PCNA）、MMP-2、TIMP-2，原位杂交法检测p27^kip1 mRNA表达水平。结果共21头小型猪完成实验：生理盐水组、空白PLGANPs组、RPM组及RPM—PLGANPs组分别6、4、5、6头。各组间损伤-狭窄模型造成的即刻损伤程度无统计学意义，RPM—PLGANPs组第30天造影血管狭窄程度明显低于生理盐水和空白NPs组（P〈0．01）和RPM组（P〈0．05）。RPM—PLGANPs组新生内膜面积NIA、内外弹力板围绕面积比值及增生指数显著小于其他三组（均P〈0．05），而最大内膜厚度MIT、内弹力板围绕面积IELA大于其他三组（均P〈0．05）。RPM—PLGANPs组阳性区域平均积分光密度值为0．35±0．06，显著高于空白NPs组（0．12±0．05，P〈0．01）及生理盐水、RPM组（0．16±0．03和0．15±0．03，P〈0．05），MMP-2／TIMP-2比值及PCNA组化染色阳性表达指数及均低于其余三组（均P〈0．05）。结论RPM-PLGA NPs结合Dispatch^TM球囊血管腔内给药抑制损伤后小型猪冠状动脉VSMC增殖和细胞外基质生成而抑制再狭窄发生，是用于治疗血管增殖性疾患有临床前景的手段之一。

Objective To determine whether intramural administration of rapamycin （RPM）- loaded polylactic-polyglycolic acid （PLGA） nanoparticles （NPs） can reduce intimal thickening and affect the mRNA expressions of matrix metalloproteinase （MMP） -2, tissue inhibitor of metalloproteinase （TIMP） - 2 and p27^kip1 in a coronary injury-stenosis model of minipigs. Methods Twenty eight minipigs were randomly separated into four groups： saline group （n =7）, blank PLGA NPs group （5.0 mg/ml） （n =7）,RPM group （ 1.0 mg/ml ） （ n = 7 ）, and RPM-PLGA NPs （ 5. 0 mg/ml ） group （ n = 7 ）, respectively. Different treatments were intracoronary locally delivered via a Dispatch^TM catheter for 10 minutes. Serial angiography was performed pre-and post-modeling 30 days and the percent stenosis degree was assessed. Hematoxylin-Eosin （HE） staining, Weigert＇s resorcin fuchsin staining and picric acid-sirius red staining were used for morphometric analysis. Immunohistochemistry was performed to assess the levels of proliferating cell nuclear antigen （ PCNA）, MMP-2, and TIMP-2 at early and late time points, respectively. The expression of p27^kip1 mRNA was detected by in sitn hybridization staining. Results Data from 21 minipigs had been collected at the end of the experiment with 6, 4, 5, and 6 from the former mentioned 4 groups, respectively. For the instant injury index, there was no significant difference among the four groups. The percent stenosis degree of RPM-PLGA NPs group was significantly lower than that of the other three groups respectively （ all P 〈 0.05 ）. The neointima area, net external elastic lamina area to external elastic laminal area ratio, and proliferative index of RPM-PLGA NPs group were significantly less than those of the other three groups, with all the P values less than 0. 05. The mean value of integral optical density of p27^kip1 mRNA expression of RPM-PLGA group was 0. 35 ±0.06, higher than that of blank PLGA NPs group （0. 12 ±0.05, P〈 0.01）, saline group （0.16 ±0.03, P〈 0.05）, and RPM group （0.15±0.03, P〈 0.05 ）, respectively. The MMP-2/TIMP-2 ratio and the positive expression index of PCNA in RPM-PLGA group were lower than that of the other groups （ P 〈 0. 05 ）. Conclusions Locally delivered rapamyeinloaded PLGA NPs significantly reduces MMP-2/TIMP-2 ratio and PCNA expression, increases p27^kip1 mRNA expression and significantly relieves percent stenosis degree and shows excellent acute procedural results in the minipig interventional coronary artery oversized balloon injury model. The results from minipig model further support that this approach could be a potential clinical procedure for vascular proliferative disease.