大鼠颈动脉球囊损伤局部A20基因转染后内皮细胞再生及
血管超微结构的影响*☆
高 静，李 蕾，徐 丹，陈 良，曲 鹏
课题背景：锌指蛋白A20为近年来在细胞内源性保护机制的研究中发现的一类新的蛋白质，是核因子κB信号系统活化的产物，并负反馈抑制核因子κB的活化而对细胞损伤起保护作用。随着对血管内皮细胞增生分子生物学机制的深入认识和分子生物学技术的发展，基因治疗有可能成为未来解决这一问题的突破点之一。
同行评价：文章报告了A20基因转染对球囊损伤血管内皮细胞及平滑肌的影响，结果表明，A20基因转染可以改善损伤部位内皮的修复和逆转损伤引起的平滑肌细胞表型转化，可能对防治经皮冠状动脉支架植入后血管内皮细胞增生致再狭窄有一定作用，文章有一定的创新性。
相关链接：大量实验研究发现，完整的内皮层能抑制平滑肌细胞增生、迁移及细胞外基质合成。内皮剥脱造成新生内膜增厚。支架置入部位的早期内皮化可能预防血栓形成和再狭窄。研究者们曾试用多种不同方案，包括药物调控，组织工程、基因和干细胞治疗，甚至操作方法的改进（即直接置入支架）以期限制内皮损伤；补充内皮产物、加速内皮细胞再生；或三管齐下，以期减少新生内膜增厚。最为天然的途径应该还是刺激再内皮化，促进愈合过程的加速。
摘要
目的：锌脂蛋白A20是核因子κB信号系统活化的产物。研究证实核因子κB的激活是球囊损伤动脉后血管狭窄发生的重要机制之一。实验拟观察锌指蛋白A20对大鼠颈总动脉球囊损伤局部血管内皮再生和血管超微结构的影响。
方法：实验于2006-03/2007-06在大连医科大学附属第二医院中心实验室完成。①实验材料：健康雄性SD大鼠42只，体质量350~400 g。②分组及实验过程：采用随机数字表法将大鼠分为3组，每组14只。制备A20质粒DNA，建立大鼠颈总动脉球囊损伤模型（假手术组只进行颈动脉结扎，不进行球囊损伤术）；对照组：球囊损伤术后局部灌注Lipofectamine 2000 Reagent+TE缓冲液；治疗组：球囊损伤术后局部灌注Lipofectamine 2000 Reagent +TE缓冲液+pCAGGS-GFP/A20重组质粒。A20质粒DNA在球囊损伤大鼠颈动脉的局部转染。③实验评估：荧光显微镜观察A20在损伤动脉壁的转染效率；伊文思蓝染色法评估损伤后颈动脉内皮再生情况；透射电镜观察血管超微结构改变。实验过程对动物的处理符合动物论理学标准。
结果：①球囊损伤术和A20转染治疗后2周，治疗组再内皮化百分比大于对照组（P < 0.05）。②假手术组大鼠颈动脉平滑肌细胞电镜下呈典型的“收缩表型”；对照组球囊损伤后3 d可见合成型细胞及过渡型细胞，7 d血管平滑肌细胞表型发生明显改变，呈典型的“合成表型”，治疗组术后7 d可见接近收缩型的血管平滑肌细胞。
结论：局部转染A20基因可促进损伤血管内皮再生，抑制损伤处血管平滑肌细胞表型转化。
关键词：锌指蛋白A20；内皮；超微结构；基因转染；大鼠����
高静，李蕾，徐丹，陈良，曲鹏. 大鼠颈动脉球囊损伤局部A20基因转染后内皮细胞再生及血管超微结构的影响[J].中国组织工程研究与临床康复，2008，12(15):2838-2842 [www.zglckf.com/zglckf/ejournal/upfiles/08-15/15k-2838(ps).pdf]
大连医科大学附属第二医院心内科，辽宁省大连市 116027
高 静☆，女，1979年生，山东省平原县人，汉族，大连医科大学在读博士，主要从事介入心脏病学的研究。
slgaojing@sohu.
com
通讯作者：曲 鹏，博士，博士生导师，大连医科大学附属第二医院心内科，辽宁省大连市 116027
qupeng777@yahoo.
com.cn
国家自然科学基金资助项目（30670836） *
中图分类号:R329.412
文献标识码:B
文章编号:1673-8225
(2008)15-02838-05
收稿日期：2007-09-25 修回日期：2007-01-04 (07-50-9-5250/W·Q)
Abstract
AIM: Zinc finger protein A20 is the activated product of nuclear factor-κB signaling. Activation of nuclear factor-κB is one of important mechanisms of angiostegnosis after balloon injury. This study aimed to investigate the effects of in vivo local transfection of zinc finger protein A20 gene on reendothelialization and vascular ultrastructure of rats with carotid artery balloon injury.
METHODS: Experiments were performed at the Central Laboratory of Second Affiliated Hospital of Dalian Medical University from March 2006 to June 2007.①Forty-two healthy male Sprague-Dawley (SD) rats of 350-400 g were selected. ②The rats were randomly divided into three groups equally. A20 plasmid DNA was prepared. Rat models of common carotid artery balloon injury were established. Rats in the sham operation group only received ligation of carotid. Rats in the control group were administered with Lipofectamine 2000 Reagent+TE buffer solution in local region after sac injury. Rats in the treatment group were treated with Lipofectamine 2000 Reagent +TE buffer solution+pCAGGS-GFP/A20 recombinant plasmid after sac injury. A20 plasmid DNA was locally transferred into rats with carotid balloon injury. ③The transfection efficiency of A20 plasmid in injured arterial wall was evaluated by fluorescence microscope. Endothelial regeneration of corotid artery was evaluated by Evans blue dye staining. Ultrastructural change in injured artery was observed with a transmission electron microscope. The disposal in animals was accorded with the animal ethical standards.
RESULTS: ①Local transfection of A20 gene resulted in an increase in reendothelialization, as measured as percent area reendothelialized at day 14 in the treatment group compared to the control group (P < 0.05). ②Contractile phenotype of vascular smooth muscle cells (VSMCs) was shown in the sham operation group and synthetic phenotype and transition cells were shown after balloon injury in the control group at day 3. VSMCs showed typical synthetic phenotype at day 7, at the same time, VSMCs resembled contractile phenotype in the treatment group at day 7.
CONCLUSION: Local transfection of A20 gene can accelerate reendothelialization after arterial injury and inhibit the phenotype transformation of VSMCs.
Gao J, Li L, Xü D, Chen L, Qu P. Effects of zinc finger protein A20 on reendothelialization and vascular ultrastructure of rats with carotid artery balloon injury.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2008;12(15):2838-2842(China) [www.zglckf.com/zglckf/ejournal/upfiles/08-15/15k-2838(ps).pdf]
Department of Cardiology, Second Affiliated Hospital, Dalian Medical University, Dalian 116027, Liaoning Province, China
Gao Jing☆, Studying for doctorate, Department of Cardiology, Second Affiliated Hospital, Dalian Medical University, Dalian 116027, Liaoning Province, China
slgaojing@sohu.com
Correspondence to: Qu Peng, Doctor, Tutor of doctor, Department of Cardiology, Second Affiliated Hospital, Dalian Medical University, Dalian 116027, Liaoning Province, China
qupeng777@yahoo.
com.cn
Supported by: the National Natural Science Foundation of China, No. 30670836*
Received: 2007-09-25
Accepted: 2008-01-04
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