摘要
目的评价聚（乳酸-羟基乙酸）共聚物／壳聚糖（PLGA／CS）纳米粒超细纤维复合膜血管外支架缓释组织因子小干扰RNA（TFsiRNA）抑制静脉移植物内膜增生的有效性。方法使用改进静电纺丝技术制备PLGA／CS纳米粒超细纤维复合膜。氯仿溶解复合膜PLGA后测量壳聚糖含量，检测超细纤维复合膜吸水率。建立SD大鼠右颈外静脉．颈总动脉间置移植模型，随机分为5组：A组（PLGA／CS—TFsiRNA纳米粒外支架组），B组（PLGA／CS—Stealth^TMRNAi阴性对照纳米粒外支架组），C组（PLGA／CS空白纳米粒外支架组），D组（PLGA外支架组），E组（无外支架组）。BLOCK-iT^TM荧光寡核苷酸检测静脉移植物转染。分别于术后1、3、7、14、28d取标本。免疫印迹和免疫组化检测管壁组织因子蛋白表达，免疫组化检测管壁增殖细胞核抗原表达，计算机图像系统分析新生内膜厚度。结果通过调节静电纺丝PLGA和壳聚糖纳米粒溶液流量控制复合膜中PLGA和壳聚糖含量。复合膜由于加入壳聚糖具有良好吸水性。术后12d静脉移植物管壁可检测BLOCK-iTM荧光寡核苷酸绿色荧光。术后3、7d，A组组织因子蛋白表达明显低于其他组（P〈0．05，其中术后3d分别为0．40±0．03与0．75±0．01、0．75±0．05、0．77±0．07，术后7d分别为0．30±0．03与0．84±0．05、0．86±0．06、0．85±0．06），术后14d，A组增殖细胞核抗原表达明显低于其他组（P〈0．01，13．0％±2．6％与25．0％±2．8％、24．2％±3．9％、24．0％±4．1％、44．8％±3．7％），A组术后28d新生内膜厚度明显低于未治疗组，P〈0．01，（18．8±2．9）μm与（38．7±5．0）μm、（37．3±3．6）μm、（37．2±2．6）μm、（67．5±4．8）μm。结论静电纺丝制备PLGA／CS纳米粒超细纤维复合膜血管外支架缓释组织因子siRNA能有效抑制大鼠静脉移植物早期内膜增生，这将成为基因治疗静脉移植物狭窄一种新方法。
Objective To evaluate a strategy of using TF siRNA loaded in a novel external stent prepared by hybrid uftrafine fibrous membrane consisting of PLGA/Chitosan nannoparticles as a therapy for vein graft disease. Methods Hybrid ultrafine fibrous membranes consisting of PLGA/Chitosan nannoparticles were fabricated via a specially designed electrospinning setup. After soaking in chloroform to dissolve PLGA, the amount of chitosan in the hybrid membranes was determined. The water uptake of the hybrid ultrafine fibrous membranes was investigated by incubation in phosphate buffer solution. Right jugular vein-carotid artery interposition grafting models in Sprague-Dawley rats were randomly divided into five groups ： Group A （ external stent consisting of PLGA/CS-TFsiRNA nanoparticles ） , Group B （ external stent consisting of PLGA/CS-StealthTM RNAi negative control nanoparticles） , Group C（ external stent consisting of PLGA/CS blank nanoparticles） , Group D（ external stent consisting of PLGA） , Group E （without perivenous external stent）. BLOCK-iTTM Fluorescent Oligo was used to confirm its stability and successful transfer into the vein graft wall. The vein grafts were harvested at 1, 3, 7, 14, 28 d after operation, respectively. The TF protein expression of vein grafts was analyzed by Western blot and immunochemistry at 1,3,7 d after operation, respectively. The expression of proliferating cell nuclear antigen （PCNA） was identified by immunochemistry methods. The thickness of neointima at 28 d was calculated by computer imaging analysis system. Results The PLGA and CS amount in PLGA/Chitosan nannopartieles membranes could be well controlled by adjusting the flow rate for electrospinning of PLGA and chitosan nannoparticles, respectively. Because of the introduction of chitosan,which is a naturally hydrophilic polymer, the hybrid membranes exhibited good water absorption properties. BLOCK-iT^TM Fluorescent Oligo could be detected in the graft wall even 12days after operation. The expression of TF protein in Group A was significantly less than that in control groups at 3 d after operation （ P 〈 0. 05,0. 40 ±0. 03 vs 0. 75 ± 0. 01,0. 75 ±0. 05,0. 77 ±0. 07） and at 7 d after operation（P 〈0. 05,0. 30 ±0. 03 vs 0. 84±0. 05,0. 86 ±0. 06,0. 85±0. 06）. The expression of PCNA in Group A decreased significantly in comparison with control groups at 14 d after operation （P〈0.01, 13.0% ±2.6% vs 25.0% ±2.8%,24.2% ±3.9%,24.0% ± 4.1% ,44. 8%± 3.7% ）. The thickness of neointima at 28 d after grafting in Group A was significantly less than the untreated group（P〈0.01, 18.8 μm ±2.9μm vs 38.7 μm ±5.0 μm,37.3 μm±3.6 μm,37.2μm ±2.6 μm, 67. 5μm ± 4. 8 μm）. Conclusion The novel external stent prepared by hybrid ultrafine fibrous membrane consisting of PLGA/Chitosan nannoparticles inhibits early neointima formation in rat vein grafts. This strategy may be a practicable and promising form of gene delivery against vein graft failure.
出处
《中华医学杂志》
CAS
CSCD
北大核心
2009年第41期2938-2942,共5页
National Medical Journal of China
基金
国家自然科学基金（30571839）
湖北省卫生厅科研基金（JX2B16）
