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|本期目录/Table of Contents|

hVEGF165四环素调控腺病毒载体的构建及其在大鼠成肌细胞表达(PDF)

《心脏杂志》[ISSN:1009-7236/CN:61-1268/R]

期数:
2006年第1期
页码:
35-38，42
栏目:
基础研究
出版日期:
2006-01-01

文章信息/Info

Title:
Construction of tetracyclineregulative adenoviral vector containing human vascular endothelial growth factor 165 gene and its expression in rat myoblast
作者:
黄新苗1; 秦永文1; 沈茜2; 吴弘1; 陈少萍1; 周菲1; 游晓华1
第二军医大学长海医院:1.心内科，2.实验诊断科，上海 200433
Author(s):
HUANG Xin-miao1;  QIN Yong-wen1;  SHEN Qian2;  WU Hong1;  CHEN Shao-ping1;  ZHOU Fei1;  YOU Xiao-hua1
1. Department of Cardiology，2. Department of Laboratory Diagnosis, Changhai Hospital, Second Military Medical University，Shanghai 200433，China
关键词:
血管内皮生长因子; 成肌细胞; 重组腺病毒; 基因治疗
Keywords:
VEGF; myoblast; recombinant adenoviruses; gene therapy
分类号:
R331.32;Q789
DOI:
-
文献标识码:
A
摘要:
目的 构建表达人血管内皮生长因子165基因（hVEGF165）的四环素调控重组腺病毒，并测定其在原代培养大鼠成肌细胞的表达水平。方法 亚克隆hVEGF165全长cDNA到穿梭质粒pTREShuttle2，获得四环素可调控的目的基因表达盒；亚克隆表达盒到BD Adeno-XTM Viral DNA，获得重组腺病毒质粒；重组质粒转染HEK293细胞以包装产生具有感染能力的重组腺病毒颗粒AdTRE.VEGF165。hVEGF165重组腺病毒和Tetoff 调节病毒共感染原代培养的成肌细胞，酶联免疫法检测目的基因的表达。结果 成功构建了能表达hVEGF165的四环素调控的重组腺病毒载体。hVEGF165重组腺病毒(20 pfu/cell)和Tetoff 调控病毒(20 pfu/cell)共感染骨骼肌成肌细胞后5 d， ELISA检测显示培养上清中VEGF165蛋白浓度显著高于未感染的细胞培养上清［(14.80±1.15 ng/ml） ×105/(cell·24 h) vs （1.39±0.36 ng/ml）×105/(cell·24 h),P<0.01 )］。结论 四环素调控表达的腺病毒系统可高效地转导VEGF基因进入哺乳动物细胞；原代培养的骨骼肌成肌细胞感染AdTRE.VEGF165后可高水平地分泌VEGF165蛋白。
Abstract:
AIM To construct tetracycline (Tet)regulatable recombinant adenovirus containing human vascular endothelial growth factor 165 gene (hVEGF165), and to detect its expression in myoblast. METHODS A Tetresponsive mammalian expression cassette, which could be regulated by tetracycline, was made by subcloning the fulllength cDNA encoding hVEGF165 into pTREShuttle2 vector. The expression cassette was then subcloned into the BD AdenoXTM Viral DNA to form an adenoviral plasmid. Next, the recombinant plasmid was packaged into infectious recombinant adenoviral particles by transfecting HEK 293 cells. Primary rat skeletal myoblasts were infected with both the hVEGF165 recombinant adenovirus and Tetoff regulatory virus. Expression was tested by enzymelinked immunosorbent assay. RESULTS We successfully constructed a tetracycline regulatable recombinant adenoviruses vector containing the cDNA for human VEGF165, a secreted endothelial cell specific angiogenic growth factor. The primary skeletal myoblasts infected with AdTRE.VEGF165 (20 pfu/cell) and Tetoff regulatory virus (20 pfu/cell) demonstrated higher VEGF165 protein secretion into the supernatant compared with uninfected group［(14.80±1.15 ng/ml)×105/(cell·24 h) vs (1.39±0.36 ng/ml)×105/(cell·24 h), P<0.01 ］, as measured by ELISA at 5 d after infection. CONCLUSION Tetregulatable adenoviral expression systems are used to deliver gene of VEGF into mammalian cells efficiently. The primary skeletal myoblasts infected with AdTRE.VEGF165 secretes VEGF165 protein at high levels.

参考文献/References

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［7］von Degenfeld G, Banfi A, Springer ML, et al. Myoblastmediated gene transfer for therapeutic angiogenesis and arteriogenesis［J］. Br J Pharmacol, 2003, 140(4):620-626.

［8］Germani A, Di Carlo A, Mangoni A, et al. Vascular endothelial growth factor modulates skeletal myoblast function［J］. Am J Pathol, 2003, 163(4):1417-1428.

［9］郝亚荣，李庚山，李建军，等. 血管内皮生长因子对培养内皮细胞合成一氧化氮的影响［J］. 心脏杂志，2002，14(3):186-188.

备注/Memo

备注/Memo:
收稿日期: 2005-05-20.基金项目：国家自然科学基金项目资助（No.30070750）通讯作者：秦永文，主任医师，博士，主要从事心脏病的介入治疗等方面的研究 Tel:(021)25074707 作者简介：黄新苗，主治医师，讲师，硕士Tel：(021)25072146 Email：huangxinmiao@hotmail.com
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