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3讨论
缺氧诱导因子1(hypoxiainducible factor1,HIF1)于1995年在研究促红细胞生成素(erythropoietin, EPO)的过程中发现[8]。作为一种DNA结合蛋白，HIF1广泛的表达于缺氧诱导的哺乳动物细胞中。它的主要结构是异源二聚体蛋白，异源四聚体比较少见。其中的亚基分别由120kDa的α亚基和91,93,94(3种分子质量)kDa的β亚基。α亚基，即缺氧诱导因子1α(hypoxiainducible factor1α，表1不同培养条件下RF/6A细胞HIF1α的表达(n=25)表2不同培养条件下RF/6A细胞内活性氧水平(n=30) HIF1α)是该蛋白的功能性亚基，在HIF1的蛋白稳定调节方面发挥重要作用[9]。HIF1α的C端存在两个反式激活结构域(transactiviation domain, TAD)，分别相应的称为靠近N端的TADN(531575位氨基酸)和C端的TADC(786826位氨基酸)。在NTAD中含有一个重要特定的氨基酸结构，称为氧依赖的降解结构域(oxygendependent degradation domain,ODD)，是HIF1的降解位点[10,11]。HIF1的活性调节包括氧依赖和非氧依赖两种途径，氧依赖途径即通过影响其HIF1α的稳定性方式完成。缺氧条件下，HIF1α结构稳定，并与HIF1β结合构成异二聚体，形成反式作用元件，与靶基因结合，达到调控靶基因转录活性的作用。常氧条件下，脯氨酸 4羟化酶在协同因子作用下使HIF1α的ODD区发生羟基化，羟化的ODD区被VHL蛋白识别并与之结合，进而泛素化而降解[12]。而关于HIF1的非氧依赖性调节，目前的研究结果并不十分明朗。有研究认为稳定常氧下HIF1α的因素主要包括活性氧、金属离子、细胞因子、NO以及机械应力[13]。其中，对于活性氧是否稳定HIF1α，目前存在着不同的意见，部分实验结果显示活性氧不具有调节HIF1α的作用，也有应用抗氧化剂诱导肺泡上皮细胞在常氧下上调HIF1α表达的报道[1416]。相反，另一部分实验结果则显示活性氧提高HIF1α稳定性，甚至有研究提出线粒体产生的活性氧为低氧条件下保持HIF1α稳定所必须[1719]的推论。
本次实验结果显示，相对于常氧浓度，高浓度氧条件下HIF1α的表达明显增高，这与Hopf等[20]的研究部分结果相同。揭示高氧条件的促血管生成作用不仅仅是间接的导致细胞凋亡、VEGF受体下调、并形成后续常氧条件下的相对低氧，同时高氧还具有上调HIF1α的直接作用。此外，通过对活性氧组HIF1α表达的观察，发现外源性活性氧具有上调HIF1α表达的作用[21]。
通过检测活性氧水平可见，细胞内活性氧水平与其所处的氧浓度相关，与Tuttle等[22]的研究结果一致。正常细胞内产生大量活性氧分子的主要途径有：细胞进行有氧化代谢时呼吸链产生的副产物、细胞色素P450系统在发挥解毒作用时产生的ROS、呼吸爆发产物以及脂肪及其它大分子降解时的副产物。因此推测随着氧浓度的增加，细胞有氧呼吸和呼吸爆发产物增多，导致胞内活性氧水平增高。结合HIF1α表达结果，我们得出结论：相较常氧浓度条件，高氧浓度下细胞内HIF1α表达增多；外源性活性氧可增加HIF1α的表达。并给出推论：高氧浓度下HIF1α表达增高是基于胞内增多的活性氧所致。目前针对眼内新生血管拮抗药物的研究多集中于VEGF的拮抗作用，与VEGF抗体药物已介入临床使用不同，靶向HIF1拮抗的药物依然处于科研阶段[23]。因此，进一步了解高氧浓度下HIF1α表达增多的原因以及非缺氧条件下活性氧上调HIF1α表达的机制，将会为眼内新生血管的治疗(特别是早产儿视网膜病变的诊治)提供更加有效的手段和方法。
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