低氧反应元件对缺血心肌转染人血管内皮生长因子165基因表达的调控作用  

Hypoxic response elements control the expression of human vascular endothelial growth factor gene 165 transferred to ischemia myocardium

作　　者：董红燕[1] 姜波[2] 张中明[2] 王伟[2] 张宜乾[2] 徐夏红[1] 

机构地区：[1]江苏徐州医学院神经生物学研究中心,221002 [2]徐州医学院附属医院胸心外科

出　　处：《中华实验外科杂志》2008年第9期1095-1097,共3页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金资助项目（30672081）;江苏省教育厅重点资助项目（03KJA320140）

摘　　要：目的观察低氧反应元件（HRE）对低氧、复氧心肌细胞及缺血心肌转染人血管内皮生长因子165（hVEGF165）基因表达的调控作用。方法分离新生SD大鼠心肌细胞，采用不同氧条件进行培养；建立猪心肌缺血-复灌动物模型，将在HEK293T细胞包装后获得的rAVV-9HRE-hVEGF165病毒分别转染心肌细胞及动物缺血心肌。取培养心肌细胞及培养液，另取缺血区心肌组织，采用细胞免疫荧光法、酶联免疫吸附试验（ELISA）及Western blot方法分别测定hVEGF砌蛋白表达；逆转录-聚合酶链反应（RT-PCR）测定hVEGF165 mRNA表达；Ⅷ因子染色，计数缺血心肌新生血管变化。结果病毒转染率为87％；心肌细胞A、B及E组培养液中hVEGF165蛋白含量显著升高（P〈0．01），细胞免疫荧光hVEGF165蛋白染色呈阳性；RT-PCR检测显示，心肌细胞A、B及E组可见484bp目的条带；在动物整体水平，与转基因缺血组比较，转基因复灌组hVEGF165 mRNA及蛋白表达显著减弱（P〈0．01），心肌毛细血管密度亦减少。结论HRE作为氧敏感基因调控开关能有效地调控缺血心肌转染hVEGF165基因的表达。Objective To investigate the effects of hypoxic response elements （HRE） on the expression of rAVV-gHRE-hVEGF165 transferred to cultured eardiocytes and isehemia myocardium under hypoxie and reoxygenation conditions. Methods Cardiocytes of neonatal Sprague Dawley rats were cultured and incubated with rAAV-9HRE-hVEGF165 , and pig isehemie heart models were established and rAAV- 9HRE-hVEGF165 was injected into isehemia myocardium. RT-PCR, Western blot, ELISA and immunohistochemistry were used to detect the hVEGF165 expression in cultured eardiocytes and myocardium under hypoxie and reoxygenation conditions. Results RT-PCR and ELISA revealed that, in cultured eardiocytes, the expression of hVEGF165 mRNA and protein were up-regulated under hypoxie conditions. After reoxygenation, the hVEGF165 mNRA expression was decreased （P 〈 0.01 ）. RT-PCR and Western blot also showed that, under myocardial isehemia, the hVEGF165 expression was increased significantly （P 〈 0.01 ）. Following myocardial reperfusion, both hVEGF165 mRNA and protein expression was inhibited （P 〈 0.01 ）. The new vessels following the reperfusion were decreased. Conclusion 9HRE, can effectively control the hVEGF165 gene expression under altered oxygen conditions in vitro and in vivo.

关 键 词：低氧反应元件 血管内皮生长因子 心肌 基因表达 

分 类 号：R541[医药卫生—心血管疾病]