Molecular analyses of hypoxia response of pulmonary arterial endothelium by using genetic engineering

[目的]低氧诱导因子I(HW-1)最初被认为是一种由低氧诱导并与促红细胞生成素(EPO)低氧反应元件(HRE)中的某个部位结合的核因子。HIF-1通过激活基因转录在02内稳态中发挥重要作用，这些基因的产物介导必要的细胞和系统对缺氧的反应，包括红细胞生成、糖酵解、血管生成和血管扩张。本研究的目的是在腺病毒介导的Re基因和低氧反应顺式元件的共同作用下，通过对分泌报告基因(Re)产物的基因修饰来阐明肺动脉内皮细胞对低氧的反应。[方法]构建两种腺病毒载体，观察血管内皮细胞对缺氧的反应。其中一种腺病毒载体含有从EPO基因组中分离的含低氧顺式元件的Re表达盒(AdHRE)，另一种腺病毒载体含有不含…的Re表达盒更多的HRE(Ad)作为对照载体。利用这些载体，通过缺氧和常氧条件下Re产物的产生比率来评价PA内皮、主动脉内皮细胞的缺氧反应。[结果]与正常氧条件下的Ad处理的内皮细胞相比，各Ad处理的内皮细胞在低氧条件下的Re生成率无明显增加。低氧条件下AdHRE感染的PA内皮细胞的Re生成率为AdHRE感染的PA内皮细胞的4倍(p&lt；0.01)。此外，有趣的是，AdHRE感染缺氧的PA远端内皮细胞的Re生成率显著增加，是缺氧下感染AdHRE的PA近端内皮细胞的2倍(p&lt；0.01)。为了评价不同类型内皮细胞对缺氧的反应，用AdHERE处理后的主动脉内皮细胞暴露于低氧环境中。AdHRE处理的主动脉内皮细胞在缺氧12h后的Re生成率处于平台期，是常氧条件下AdHRE处理的每种内皮细胞的4到2倍。[结论]用腺病毒介导的缺氧反应顺式元件转移建立了缺氧反应分析系统。我们目前使用该HRE转移系统的研究表明，在体循环中，PAE的缺氧反应明显大于AE，尤其是与血管重塑相关的PA远端内皮细胞的缺氧反应明显更大。该HRE基因转移系统可用于评价循环系统或肺部疾病的缺氧反应障碍。较少

[Purpose] Hypoxia-inducible factor I (HW-1) is originally identified as a nuclear factor that is induced by hypoxia and bound to a site in the erythropoietin (Epo) hypoxia-response element (HRE). HIF-1 plays an important role in 02 homeostasis by activating transcription of genes whose' products mediate essential cellular and systemic response to hypoxia, including erythropoiesis, glycolysis, vasculogenesis, and vasodilatation. The aim of this study is to clarify hypoxia response of pulmonary arterial (PA) endothelium by addition of genetic modification to secrete reporter gene (Re) product following adenovirus-mediated transfer of both Re cDNA and hypoxia response cis-element. [Method] To evaluate hypoxia response of PA endothelium, two type of adenovirus vector were constructed. One adenovirus vector contains Re expression cassette with hypoxia response cis element (HRE) isolated from Epo genome (AdHRE), and the other one is adenovirus vector containing Re expression cassette without … More HRE (Ad) as control vector. Using these vectors, hypoxia response of PA endothelium, aortic endothelium was evaluated by using the ratio of production of Re product in exposure to hypoxia and normoxia. [Results) Each Ad treated-endothelium exposed to hypoxia had no increase in the ratio of Re production, compared with Ad treated endothelium in normoxia. In marked contrast, AdHRE-infected PA endothelium exposed to hypoxia had an increase in the ratio of Re production, 4 fold compared with AdHRE infected PA endothelium in normoxia (p<O.OOl). Furthermore, interestingly, AdHRE infected endothelium in distal PA exposed to hypoxia had a significantly large increase in the ratio of Re production, 2 fold compared with AdHRE infected endotbeliuxn in proximal PA exposed to hypoxia (p<O.OOl). To evaluate hypoxia response in different type of endothelium, aortic endothelium was exposed to hypoxia following treatment with AdHERE.AdHRE-treated-aortic endothelium has a plateau in the ratio of Re production 12 hrs following the exposure to hypoxia, 4 or 2 fold compared with AdHRE-treated each endothelium in normoxia. [Conclusion] Analysis system of hypoxia response was established by using adenovirus-mediated transfer of hypoxia response cis element. Our current study using this HRE transfer system demonstrated that hypoxia response of PAE showed larger than that of AE of systemic circulation, especially endothelium in distal PA relevant to vascular remodeling had a markedly larger hypoxia response. This HRE- gene transfer system was feasible to evaluate disorder of hypoxia response in circulatory or pulmonary diseases. Less