Hypoxic response elements and Tet-On advanced double-controlled systems regulate hVEGF_(165) and angiopoietin-1 gene expression in vitro  

作　　者：Hao Zhang Hongyan Dong Bo Jiang Zheng Wang Rui Chen Zhifeng Zhang Zhongming Zhang 

机构地区：[1]Institute of Cardiovascular Disease, Affiliated Hospital of Xuzhou Medical College, Xuzhou, Jiangsu 221002, China [2]Center of Biological Research, Xuzhou Medical College, Xuzhou, Jiangsu 221002, China.

出　　处：《The Journal of Biomedical Research》2011年第3期204-212,共9页生物医学研究杂志（英文版）

基　　金：supported by a grant from the National Natural Science Foundation of China (No.30672081)

摘　　要：Angiogenesis in ischemic tissue is a complex and multi-gene event. In the study, we constructed hypoxic re-sponse elements （HRE） and the Tet-On advanced double-controlled systems and investigated their effects on the expression of hVEGF165 and angiopoietin-1 （Ang-1） genes in rat cardiomyocytes exposed to hypoxia and pharma-cologic induction. We infected neonatal rat cardiomyocytes with recombinant rAAV-rtTA-Rs-M2/rAAV-TRE-Tight-Ang-1 and rAAV-9HRE- hVEGF165. Our results indicated that the viral titer was 1×1012 vg /mL and the viral purity exceeded 98%. hVEGF165 expression was induced by hypoxia, but not by normoxia （P 0.001）. Ang-1 expression was evident under doxycycline induction, but undetectable without doxycycline induction （P 0.001）. Immunofluorescence staining showed that positively stained hVEGF165 and Ang-1 protein appeared only under both hypoxia and doxycycline induction. We demonstrate here that HRE and the recombinant Tet-On advanced double gene-controlled systems sensitively regulate the expression of hVEGF165 and Ang-1 genes in an altered oxygen environment and under pharmacological induction in vitro.Angiogenesis in ischemic tissue is a complex and multi-gene event. In the study, we constructed hypoxic re-sponse elements （HRE） and the Tet-On advanced double-controlled systems and investigated their effects on the expression of hVEGF165 and angiopoietin-1 （Ang-1） genes in rat cardiomyocytes exposed to hypoxia and pharma-cologic induction. We infected neonatal rat cardiomyocytes with recombinant rAAV-rtTA-Rs-M2/rAAV-TRE-Tight-Ang-1 and rAAV-9HRE- hVEGF165. Our results indicated that the viral titer was 1×1012 vg /mL and the viral purity exceeded 98%. hVEGF165 expression was induced by hypoxia, but not by normoxia （P 0.001）. Ang-1 expression was evident under doxycycline induction, but undetectable without doxycycline induction （P 0.001）. Immunofluorescence staining showed that positively stained hVEGF165 and Ang-1 protein appeared only under both hypoxia and doxycycline induction. We demonstrate here that HRE and the recombinant Tet-On advanced double gene-controlled systems sensitively regulate the expression of hVEGF165 and Ang-1 genes in an altered oxygen environment and under pharmacological induction in vitro.

关 键 词：gene control vascular endothelial growth factor ANGIOPOIETIN-1 hypoxia DOXYCYCLINE CARDIOMYOCYTE 

分 类 号：Q785[生物学—分子生物学] TP242[自动化与计算机技术—检测技术与自动化装置]