中国组织工程研究

中国组织工程研究 ›› 2012, Vol. 16 ›› Issue (15): 2704-2708.doi: 10.3969/j.issn.1673-8225.2012.15.010

• 血管组织构建 vascular tissue construction • 上一篇    下一篇

六氟化硫微泡介导人脂联素基因兔主动脉内转染及表达☆

杨文凯1, 2，陈  庆3，杨春丽4，王现国1，谢  霆1，董念国1，陈新忠1   

  1. 华中科技大学同济医学院附属协和医院，1心外科；3普外科，湖北省武汉市  430022；江西省人民医院，2胸心外科；4 ICU，江西省南昌市  330006
  • 收稿日期:2011-12-21 修回日期:2012-01-27 出版日期:2012-04-08 发布日期:2012-04-08
  • 通讯作者: 陈新忠，博士，主治医师，华中科技大学同济医学院附属协和医院心外科，湖北省武汉市 430022 xinzhongchen@yahoo.com.cn
  • 作者简介:杨文凯☆，男，1975年生，山东省新泰市人，汉族，1975年生，华中科技大学同济医学院在读博士，主治医师，主要从事心外科临床和基础研究方面的研究。doctor_wenkaiyang@yahoo.com.cn

Transfection and expression of human apM1 gene into the aorta of rabbits mediated by using ultrasound with SonoVue   

Yang Wen-kai1, 2, Chen Qing3, Yang Chun-li4, Wang Xian-guo1, Xie Ting1, Dong Nian-guo1, Chen Xin-zhong1   

  1. 1Department of Cardiosurgery, 3Department of General Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan  430022, Hubei Province, China; 2Department of Cardiothoracic Surgery, 4ICU Department, Jiangxi Provincial People’s Hospital, Nanchang  330006, Jiangxi Province, China
  • Received:2011-12-21 Revised:2012-01-27 Online:2012-04-08 Published:2012-04-08
  • Contact: author: Chen Xin-zhong, Doctor, Attending physician, Department of Cardiosurgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, Hubei Province, China xinzhongchen@yahoo.com.cn
  • About author:Yang Wen-kai☆, Studying for doctorate, Attending physician, Department of Cardiosurgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, Hubei Province, China; Department of Cardiothoracic Surgery, Jiangxi Provincial People’s Hospital, Nanchang 330006, Jiangxi Province, China doctor_wenkaiyang@yahoo.com.cn

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摘要：

背景：超声微泡载基因传输技术是一种新兴起的定向传输基因技术。
目的：构建含人脂联素基因脂联素的真核表达载体，采用六氟化硫微泡介导脂联素基因进行兔主动脉转染及表达，为脂联素用于体内干预动脉粥样硬化的研究奠定基础。
方法：人心外膜脂肪组织提取总RNA构建pcDNA3.1-脂联素重组体，通过瞬时转染人脐静脉内皮细胞验证载体构建是否成功。21只大白兔随机分成对照组(n=6)和脂联素转基因组(n=15)，脂联素转基因组经兔耳缘静脉注射pcDNA3.1-脂联素与六氟化硫微泡的混合物，诊断超声仪照射兔胸腹主动脉区域，于照射后2，7，14 d取兔主动脉血管及血清，检测主动脉血管壁中脂联素基因的表达及血清中脂联素蛋白水平。
结果与结论：pcDNA3.1-脂联素瞬时转染入人脐静脉内皮细胞可有效表达。六氟化硫微泡传输脂联素基因2 d后即可检测到兔主动脉血管壁脂联素基因高表达，持续至14 d，且兔血清脂联素蛋白水平显著增加(P < 0.01)。说明pcDNA3.1-脂联素重组体构建成功，六氟化硫微泡可在快速血流状态下，安全、高效传输脂联素基因入兔主动脉血管壁并有效表达与分泌至血浆。
关键词：六氟化硫微泡；脂联素；动脉粥样硬化；基因；重组质粒
doi:10.3969/j.issn.1673-8225.2012.15.010

关键词: 六氟化硫微泡, 脂联素, 动脉粥样硬化, 基因, 重组质粒

Abstract:

BACKGROUND: Gene delivery using ultrasound microbubbles is an emerging directional transmission gene technology.
OBJECTIVE: To construct a recombinant plasmid containing human apM1 gene and delivery apM1 gene into the aorta of rabbits by using ultrasound with SonoVue. 
METHODS: Twenty-one rabbits were randomized into control group (n=6) and apM1 group (n=15). apM1 mRNA from epicardial adipose tissue was amplified by reverse transcription-PCR and cloned into plasmid pcDNA3.1 to generate the recombinant plasmid pcDNA3.1-apM1, which was confirmed by nucleotide sequencing and transiently transfected into human umbilical vein endothelial cells (HUVECs). While the mixture of SonoVue and pcDNA3.1-apM1 was injected though helix vein, the thoracoabdominal aorta of rabbits were sonicated by ultrasound for 3 minutes. The arterial vessels and blood serum of rabbits were harvested at 2, 7and 14 days respectively after delivery to detect apM1 expression by Western blot analyses and enzyme linked immunosorbent assay (ELISA).
RESULTS AND CONCLUSION: The identification of pcDNA3.1-apM1 recombinant by DNA sequencing ws correct. The expression of apM1 gene could be detected by Western blot in HUVECs after pcDNA3.1-apM1 transient transfection. apM1 gene could be effectively transfered into the aorta wall of rabbit by using ultrasound with SonoVue. The high expression of apMl gene was detected at 2 days after delivery and until to 14 days. ELISA results showed that the concentration of apM1 protein in serum of rabbit could be significantly increased by delivery of apM1 gene using ultrasound with SonoVue (P < 0.01). These findings indicate that the eukaryotic expression recombinant containing apM1 gene can be successfully constructed. apM1 gene can be transferred into the deep area of rabbit aorta vessel wall by ultrasound with SonoVue and expressed and secreted effectively. 

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