壳聚糖纳米粒子携带反义增殖细胞核抗原寡核苷酸抑制血管移植后内膜增殖的实验研究

INHIBITION OF INTIMAL PROLIFERATION AFTER VEIN GRAFTING BY CHITOSAN NANOPARTICLE WITH PROLIFERATION CELL NUCLEAR ANTIGEN-ANTISENSE OLIGO DEOXY NUCLEOTIDES

目的探讨壳聚糖纳米粒子携带反义基因在血管外科领域中的应用。方法雄性SPF级SD大白鼠54只,体重450～600g,随机分为实验组和对照组(n=27)。实验组取右侧颈静脉、颈动脉吻合口段灌注携带增殖细胞核抗原(proliferation cell nuclear antigen,PCNA)的反义寡核苷酸(antisens oligo deoxy nucleotides,ASODN)粒子后,移植至同侧颈动脉;对照组用同样方法灌注生理盐水。取造模后血管通畅的48只大白鼠(n=24)分别于第1、2、3、4周超声多普勒监测血流动力学,取标本行免疫组织化学染色、Western blot蛋白印迹检测、血管壁组织病理变化检测等。结果两组的血栓形成率差异无统计学意义(P>0.05)。在4周的观察期内,代表增殖的PCNA蛋白印迹条带显示:实验组移植静脉、吻合口的浓度较对照组低。术后1、2、3及4周,实验组移植静脉PCNA阳性细胞指数分别为0.13%±0.11%,0.79%±0.28%,0.45%±0.29%,0.43%±0.25%,吻合口分别为1.90%±0.84%,2.11%±0.98%,2.48%±0.77%,2.17%±0.36%,较对照组明显减少(P<0.05);实验组移植静脉及吻合口的空腔面积分别为88.71±16.96、95.98±21.44、88.48±32.81、97.86±34.11μm2和41.49±3.34、45.15±11.65、46.27±8.90、51.62±8.85μm2,均较对照组大(P<0.05),实验组移植静脉、吻合口的内膜面积/中膜面积分别为22.73%±3.11%、32.40%±4.55%、45.14%±3.19%、45.70%±5.01%和41.49%±3.34%、45.15%±11.65%、46.27%±8.90%、51.62%±8.85%,均较对照组小(P<0.05)。最大血流速度比较,两组在第1周血流最大速度相似,后3周出现不同变化。对照组静脉移植段和吻合口的最大流速逐渐增加,在第3周达到最高,至第4周时降低;实验组静脉移植段和吻合口最大流速逐渐降低,在第3周达到最低,至第4周时升高。第4周实验组和对照组移植静脉段和吻合口最大流速均接近。各时间点两组内比较差异有统计学意义(P<0.05),组间比较差异无统计学意义(P>0.05)。结论壳聚糖纳米粒子携带PCNA-ASODN能有效抑制血管移植后内膜细胞增殖,从而抑制新生内膜过度增厚。

Objective To investigate an inhibitive effect of the chitosan nanoparticles with the proliferation cell nuclear antigen （PCNA）-antisense oligo deoxy nucleotides （ASODN） on the intimal cell proliferation after the vein grafting. Methods Fifty-four male SD rats, weighing 450-600 g, were randomly divided in the experimental group and the control group of 27 rats each. In the experimental group, the chitosan nanoparticles with PCNA-ASODN were infused into the anastomosis segment of the right jugular artery and vein; then, the anastomosis segment was transplanted to the jugular artery on the same side. The rats in the control group were infused with normal saline by the same procedures. There were 24 rats in each group which used to experiment. The hemodynamic data were obtained from the Doppler ultrasound examinations at 1, 2, 3 and 4 weeks. The specimens were taken. Immunohistochemistry, Western-blot, and blood-vessel-wall histopathology were performed at the different week points. Results There was no significant difference in the thrombogenesis rate between the experimental group and the control group （3/27 vs. 3/27, P〉0. 05）. During the 4-week observation, PCNA Western-blot showed that the PCNA level was lower in the grafted vein and the anastomosis segment in the experimental group than in the control group. The indexes of the PCNA-postive proliferating cells in the intimal area （0.13%±0.11%, 0. 79%±0. 28%, 0.45± 0.29%, 0.43%± 0.25%） and the medial area （1.90%±0.84%,2.11%±0.98%,2.48±0.77%,2.17%±0-36%） were significantly decreased at 1,2,3 and 4 weeks in the experimental group when compared with those in the control group（P〈0.05）. The lumen areas in the grafted vein （88.71± 16.96, 95.98±21.44,88.48±32.81,97.86±34.11μm^2） and the anastomosis segment （41.49±3.34,45.15± 11.65,46.27±8.90,51.62±8.85 μm^2） were significantly greater in the experimental group than in the control group （P〈0.05）. The ratios of the initmal area to the medial area in the grafted vein （22.73%± 3.11%, 32.40%±4.55%, 45.14%±3.19%, 45.70%± 5.01%） and the anastomsis segment （41.49%±3.34%, 45.15%± 11.65%, 46.27%± 8.90% 51.62%±8.85%） were significantly smaller in the experimental group than in the control group（P〈0. 05）. The maximum velocities （Vmax） of the blood flow in the grafted vein and the anastomsis segment were almost the same in the two groups at 1 week, but had different changes at the next 3 week-points. In the control group, the Vmax of the blood flow gradually increased and at 3 weeks it reached the peak point; however, at 4 weeks it decreased. In the experimental group, the Vmax of the blood flow gradually decreased, and at 3 weeks it decreased to the lowest point ; however, at 4 weeks it increased. So, at 4 weeks the Vmax of the blood flow in the grafted vein and the anastomsis segment was almost the same in the two groups. There was no significant difference in the Vmax of the blood flow between the two groups （P〉0.05）, but in the same group there was a significant difference at the different time points. Conclusion The chitosan nanoparticles with PCNA-ASODN can effectively inhibit the intimal cell proliferation after the grafting of the blood vessel, so that the neointimal thickening can be prevented.