明胶联合VEGF内衬血管壁防止股动脉血管成形术后狭窄的实验研究

1、明胶联合VEGF内衬血管壁防止股动脉血管成形术后狭窄的实验研究         08-07-15 10:22:00     编辑：studa20             作者：蒋鸥 王志刚 乔正荣 时德【摘要】目的：评价血管重建术后内衬明胶和内皮细胞生长因子（VEGF）受损血管壁对防止血管狭窄的效果。方法：建立兔股动脉再狭窄动物模型，实验组内衬浓度为30%的

2、明胶及VEGF于损伤段动脉壁，分别于2周和4周采集标本，行图像分析及TUNEL原位杂交，比较实验组与对照组内膜面积（NEA）、内中膜面积比（NEA/MA）和平滑肌细胞凋亡率，电镜观察平滑肌细胞的增生及凋亡情况。结果：对照组较实验组新生内膜厚，而且细胞数也明显多。对照组NEA2周及4周分别为（1.02±0.41）和（1.46±0.15）mm2比实验组分别为（0.57±0.15）和(1.18±0.36)mm2大。NEA/MA对照组2周及4周分别为（0.54±0.14）和(0.75±0.10)也明显高于实验组分别为(0.35±0.

3、05)和（0.62±0.25）。TUNEL阳性细胞率对照组2周为（13.6±2.4）%,4周为（15.1±2.1）%，治疗组分别为（18.2±5.8）%和（17.0±1.5）%。电镜观察发现对照组平滑肌细胞明显增生，细胞器发达，肌丝较多，而实验组见较多的凋亡小体有髓样结构形成。结论：提示明胶联合VEGF内衬血管成形术后的血管内壁对血管再狭窄有明显的抑制作用。    【关键词】血管狭窄明胶内皮细胞生长因子内皮，血管兔     Investigation of vascular

4、wall lined by Glutin and VEGF to prevent rabbit femoral arteries from restenosis after angioplasty     【ABSTRACT】Objective:To evaluate the effectiveness that glutin and VEGF line the injured vasucler wall to prevent the drteries from restenosis.Methods:To build animal model of re

5、stenosis by balloon dilation in femoral artery.30 percent of glutin and VEGF were lined on injured vascular wall in the experiment,and samples were collected at 2 weeks and 4 weeks respectively.By imagine analysis system and DuTP nick end labeling,to compare neointimal area and neointimal area / med

6、ia area of the control group with those of the experment group.The hyperplasia and the apoptosis of the vascular smooth muscle cells were obsenved by electron microscopy.Results:Intima of the control was thicker thanthe experiment group,and the control group had the more number of cells than the exp

7、eriment group.From the morphology,neointimal area were reduced significantly in the experiment group(which was 0.67±0.25mm2 at the 2 weeks and 1.18±0.36 mm2 at the 4 weeks respectively)compared with the control group(which was 0.67±0.25 mm2 at the 2 weeks and 1.18±0.36 mm2 at the

8、 4 weeks).Furthermore,NEA/MA of the experimetn group(which was 0.35±0.05 at 2 weeks and 0.62±0.25 at 4 weeks)was lower than that of the control(which was 0.54±0.14 at 2 weeks and 0.75±0.10 at 4 weeks).The experiment group had the higher ratio of positive cells marked by TUNEL(whi

9、ch was 13.6±2.4% at 2 weeks and 15.1±2.1% at 4 weeks)than the control group(which was 18.2±5.8% at the 2 weeks and 17.0±1.5% at 4 weeks).Conclusion:It is effective to prevent artery from restenosis that glutin lines the injured vasucler wall.    【KEY WORDS】Restenos

10、isGelatinEndothelial growth factorsEndothelium,vascularRabbits    目前各种血管成形术后再狭窄率达30%40%1，严重地影响手术的远期效果。目前对再狭窄的研究正转向促进血管内皮细胞的修复和功能恢复2。本实验采用明胶及血管内皮细胞生长因子（VEGF）联合内衬血管内膜，观察对促进血管内皮细胞的修复和功能恢复的作用，为防止和减轻血管重建术后的再狭窄提供治疗依据。    1  材料与方法    1.1  实验动物及分组 

11、 成年日本大白兔40只,体重2.53.0kg。采用完全随机分组设计，每组10只动物。分为对照组A（2周取材）、对照组B（4周取材）、实验组A（2周取材）和实验组B（4周取材）。对照组为球囊扩张组，实验组为球囊扩张+明胶内衬组。采用10%异戊巴比妥钠1.5ml/kg加氯氨酮5mg/kg麻醉动物。    1.2  主要试剂  VEGF10g(PeproTech EC Ltd.),TUNEL检测试剂盒（武汉博士德公司）。    1.3  实验方法    1.4  标本采集

12、与制作  实验动物喂养2周及4周后取材。切取缝线下端约5cm动脉标本，采用1%PPS平衡液冲洗血管腔内残留血液，石蜡包埋，切片，HE染色，弹力纤维染色，TUNEL原位杂交，计算机图像分析，电镜观察。    1.5  损伤血管细胞凋亡的计算  正常血管内膜细胞核呈蓝色，而凋亡血管内膜细胞核呈棕黄色，在光学显微镜下计算5个高倍视野（×400）中VSMCs（血管平滑肌细胞）凋亡细胞占该视野VSMCs总数的比例，求其均值。    1.6  统计学方法  实验数据进行两样本均数间的t检

13、验，计量资料用x±s表示，率的比较用2检验。P0.05为差异有统计学意义。    2  结  果    2.1  计算机图像分析  见表1。表1  新生内膜面积（NEA）、新生内膜/中膜面积比（NIA/MA）对照组2.2  TUNEL染色结果  见表2。表2  TUNEL阳性细胞率结果提示明胶联合血管内皮细胞生长因子内衬血管壁促进了血管平滑肌细胞凋亡。    2.3  电镜观察  透射电镜可见实验组平滑肌细胞出现较多的空泡（凋亡早期改变）及凋亡小体，核膜髓样结构形成（图1A上），胞质浓缩，核有碎裂（图1A下）。对照组VSMC呈收缩型或增生型胞核大染色质丰富，胞质内肌丝较多，线粒体高尔基体发达（图1B）。    3  讨  论    PTA、经皮