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低氧反应元件调控下h--VEGF_(165)基因表达及其蛋白产物的延迟消失

张宜乾, 张中明, 闫英群, 董红燕

徐州医学院附属医院胸心外科.江苏,徐州 221002；徐州医学院神经生物研究中心.江苏,徐州 221002

摘要

血管内皮生长因子(vascular endothelial growth factor,VEGF)转基因可促进心肌缺血区血管生成,改善心脏功能,然而长期高水平表达又会引起诸多副作用。为调控VEGF表达,在启动子区加入低氧反应元件(hypoxic response element．HRE)作为调控开关,研究氧环境对VEGF基因mRNA及蛋白产物表达的影响。重组腺相关病毒(recombinant adeno--associated virus,rAAV)作为载体(rAAV--HRE--h--VEGF_(165)),转染离体培养大鼠心肌细胞,在常氧/缺氧(氧浓度1%)/缺氧复氧条件下进行培养,用酶联免疫特异性测定(enzyme linked immunosorbent assay,ELISA)方法检测培养液h--VEGF_(165)蛋白浓度,细胞免疫荧光染色观测细胞内h--VEGF_(165)蛋白表达,逆转录聚合酶链反应(reverse transcription polymerase chain reaction,RT--PCR)方法检测细胞h--VEGF_(165)mRNA表达。结果显示：未转染组、常氧培养组均无h--VEGF_(165)mRNA及其蛋白表达：缺氧培养组h--VEGF_(165)mRNA及蛋白均表达；缺氧复氧4h组的h--VEGF_(165)mRNA消失,培养液中h--VEGF_(165)蛋白减少,细胞内h--VEGF_(165)蛋白存在：缺氧复氧8h及12h组的h-VEGF_(165)mRNA消失,培养液和细胞内h--VEGF_(165)蛋白均消失。研究表明,在HRE调控下,缺氧可促使h--VEGF_(165)基因表达,复氧后,h--VEGF_(165)mRNA表达停止,蛋白产物延迟消失。

关键词： 血管内皮生长因子; 低氧反应元件; 心肌细胞

Delayed disappearance of h--VEGF_(165) mRNA and protein under regulation of hypoxic response element

Zhang Yiqian, Zhang Zhongming, Yan Yingqun, Dong Hongyan

Department of Cardiothoracic Surgery, Affiliated Hospital,Xuzhou Medical College.Xuzhou 221002,Jiangsu；China

Abstract

Transfer of vascular endothelial growth factor (VEGF) gene to ischemic myocardium may provide a useful approach for angiogenesis and improve cardiac performance. However, uncontrolled expression of VEGF in vivo may result in certain side effects, such as hemangioma formation, retinopathy, and tumor development. We investigated the feasibility of using the nine copies of hypoxic response elements (HRE) to control the expression of human VEGF_(165)(h-VEGF_(165)) under anoxic condition in cell level and also observe the synchron of both h-VEGF_(165) protein and h-VEGF_(165) mRNA expressions. Recombinant adeno-associated viral (rAAV) vector was prepared by using the three-plasmid system and cotransfected to human embryo kidney 293 T cells by the calcium phosphate precipitates method. The rAAV vector was purified by chloroform-PEG8000/NaCl -chloroform and added to cultured myocardiocyte. Myocardiocytes of Sprague-Dawley rat were cultured in serum-free medium and then randomly divided into eight groups. Group I: cultured under normoxic conditions (21% O_(2)) for 8 h as control; Group II: cultured under anoxic conditions (1% O_(2)) for 8 h; Group III: cultured under normoxic conditions (21% O_(2)) for 8 h with gene transfer; Group IV: cultured under anoxic conditions (1% O_(2)) for 8 h with gene transfer; Group V, VI, VII: cultured under anoxic conditions (1% O_(2)) for 8 h with gene transfer and then tured to normoxic conditions (21% O_(2)) for 4, 8 or 12 h, respectively; Group VIII: cultured under anoxic conditions (1% O_(2)) for 20 h with gene transfer. After completion of cell culture, the amount of h-VEGF_(165) protein in culture supernatant was quantified by using enzyme linked immunosorbent assay (ELISA). Expression of h-VEGF_(165) protein in cultured- cardiacmyocytes was also evaluated by immunofluorescence. RT-PCR was employed to detect the expression of h-VEGF_(165) mRNA. The results revealed that there were no expressions of h-VEGF_(165) mRNA and h-VEGF_(165) protein in Group I, II, III, VI and VII. After gene transferred, the expressions of h-VEGF_(165) protein and h-VEGF_(165) mRNA were significantly higher in Group IV and VIII than those in orther groups (P<0.01); Immunofluorescence positive cells were observed in Group IV, V and VIII. RT-PCR revealed that a 484 bp strip can be found in Group IV and Group VIII, but unavailable in other groups. We conclude that HRE is a promising regulator for h-VEGF_(165) gene expression following the changes of oxygen environment. HRE can induce the expression of h-VEGF_(165) gene after hypoxia, but in normal oxygen condition, the expression of h-VEGF_(165) was inhibited. Although expression of h-VEGF_(165) mRNA ceased in normal oxygen condition under the control of HRE, expression of h-VEGF_(165) protein was hysteretic to h-VEGF_(165) mRNA expression.

Key words: Vascular endothelial growth factor;hypoxic response element;cardiacmyocyte

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引用本文：

张宜乾, 张中明, 闫英群, 董红燕. 低氧反应元件调控下h--VEGF_(165)基因表达及其蛋白产物的延迟消失[J]. 生理学报 2006; 58 (3): .

Zhang Yiqian, Zhang Zhongming, Yan Yingqun, Dong Hongyan. Delayed disappearance of h--VEGF_(165) mRNA and protein under regulation of hypoxic response element. Acta Physiol Sin 2006; 58 (3): (in Chinese with English abstract).