重组腺病毒Ad-hBNP的构建及其在CHF大鼠中的分布与表达

基础医学与临床 ›› 2012, Vol. 32 ›› Issue (1): 31-35.

重组腺病毒Ad-hBNP的构建及其在CHF大鼠中的分布与表达

宋衍秋¹,赵莉莉²,²,毛用敏²,赵鸿铭¹,²,徐美林²,²,崔让庄²

1. 天津市胸科医院
2.

收稿日期:2010-12-03 修回日期:2011-06-09 出版日期:2012-01-05 发布日期:2011-12-28
通讯作者: 宋衍秋 E-mail:huozhebulei@medmail.com.cn
基金资助:
天津市卫生局项目

Construction of recombinant adenovirus Ad-hBNP and observing the distribution and the expression of hBNP gene in CHF rats

Received:2010-12-03 Revised:2011-06-09 Online:2012-01-05 Published:2011-12-28

摘要/Abstract

摘要： 目的构建重组腺病毒Ad-hBNP，观察其在CHF大鼠体内的分布与表达，为腺病毒介导的BNP治疗慢性心力衰竭奠定实验基础。方法 RT-PCR方法从人心肌组织获得hBNP片段，应用AdEasy缺陷性腺病毒载体系统，同源重组获得pAdEasy-hBNP，重组成功的pAdEasy-hBNP经阳离子脂质体法转染293T细胞，经过包装、扩增和纯化后，获得可用于实验性基因治疗的重组腺病毒Ad-hBNP；CHF大鼠24只，18只单次腹腔注射Ad-hBNP后，随机均分为1天组、4天组和7天组；另6只CHF大鼠不予以Ad-hBNP，为0点组。各实验组动物于相应的时间处死，ELISA检测血清hBNP水平，免疫组化检测CHF大鼠各脏器hBNP的表达，RT-PCR检测各组织hBNP mRNA的表达。结果应用Ad-Easy缺陷性腺病毒载体系统成功构建用于实验性基因治疗的重组腺病毒Ad-hBNP，纯化后Ad-hBNP滴度达到4.4275 ×1012VP/ml。透射电镜观察Ad-hBNP呈多面体结构，颗粒直径在90nm左右。单次腹腔注射Ad-hBNP，CHF大鼠心脏、肝脏、肺脏、肾脏、脾脏及小肠均检测到hBNP表达，外源性hBNP在CHF大鼠体内的表达至少可持续7天。结论应用Ad-Easy缺陷性腺病毒载体系统可成功构建重组腺病毒Ad-hBNP，腺病毒介导的外源hBNP基因可在CHF大鼠多脏器中得到有效表达，其生物学效应时间显著长于BNP的生物半衰期。

关键词: 脑钠肽, 腺病毒, 慢性心力衰竭, 基因治疗

Abstract: Objective To construct Ad-hBNP and observe the distribution and the expression of hBNP gene in CHF rats. The study could provide a reference of basic research for BNP in gene therapy. Methords The target gene hBNP were obstained by reverse transcription polymerase chain reaction from human heart. The pAd-hBNP were constructed through AdEasy system. Recombinant plasmid pAd-hBNP were packaged and amplified in 293T cells. Ad-hBNP used in gene therapy were acquired. 24 CHF rats were randomly divided into 4 groups for detecting the serum levels of hBNP with ELISA, the expression of hBNP with RT-PCR and immunohistochemistry in different groups. Results Ad-hBNP were constructed successfully and the titer of purified Ad-hBNP was 4.4275×1012VP/ml. Recombinant adenoviruses were showed as polyhedron shape under electron microscopy. The expression of hBNP gene were detected in CHF rats by intraperitoneal injection Ad-hBNP and it continued at least 7 days. Conclusions Ad-hBNP were constructed successfully by using AdEasy system. Exogenous hBNP were effectively expressed in CHF rats. Compared with the half life of BNP, the prosses that at least continued 7 days were significantly extended.

Key words: Brain natriuretic peptide, Adnovirus, Chronic heart failure, Gene therapy

宋衍秋赵莉莉毛用敏赵鸿铭徐美林崔让庄. 重组腺病毒Ad-hBNP的构建及其在CHF大鼠中的分布与表达[J]. 基础医学与临床, 2012, 32(1): 31-35.

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