【作者】 牟萍；

【导师】 郑荣梁；

【作者基本信息】 兰州大学 ， 细胞生物学， 2007， 博士

【摘要】 血管生成是以内皮细胞活化、细胞外基质降解、内皮细胞迁移、增殖、最终形成血管样结构为主要特征、受多种因素调节的、复杂的生理和病理过程，在此过程中活性氧（reactive oxygen species，ROS）发挥着重要作用。活性氧是细胞有氧代谢的产物，大量的活性氧会引起包括癌症在内的多种疾病，而人们对适量活性氧的认识已经从过去错误地定性为“毒性分子”重新评价为“信号分子”了。活性氧在肿瘤发生及血管生成中的信号传导作用日益受到重视，肿瘤发生、血管生成过程中ROS的来源也已成为研究的热点，其中NADPH氧化酶被认为是血管内皮细胞产生ROS的重要酶。烟酰胺腺嘌呤二核苷酸（磷酸）氧化酶（nicotinamide adenine dinucleotide（phosphate）oxidase，NADPH oxidase）是一种吞噬细胞的专有酶系，负责呼吸爆发时产生活性氧来杀死入侵的病原微生物。但从上世纪80年代中期开始，发现一些非吞噬细胞，如内皮细胞、平滑肌细胞、神经细胞等也有NADPH氧化酶同系物的表达。血管NADPH氧化酶（Vascular NADPH oxidases）是动脉粥样硬化及糖尿病性血管系统疾病中活性氧的主要来源。为了阐明ROS在血管生成中的作用及其可能的机理，我们研究了外源性H₂O₂对内皮细胞的增殖、迁移和鸡胚尿囊绒毛膜血管生成的影响以及内皮细胞NADPH氧化酶在此过程中的作用，同时观察了苯丙素甙类（phenylpropanoid glycosides）天然抗氧化剂马先蒿甙G对血管生成、肿瘤形成及生长的影响和作用机理。结果表明：1．活性氧成员之一H₂O₂，在低浓度时（10μmol／L）可以显著促进内皮细胞的增殖和迁移；但当浓度增加时，增殖和迁移受到抑制。可见H₂O₂对内皮细胞的增殖和迁移这些血管生成指标呈现出低浓度促进、高浓度抑制的双向调节作用。低浓度的H₂O₂（低于100μmol／L）对内皮细胞凋亡没有显著影响，200μmol／L H₂O₂最大程度地诱导内皮细胞凋亡，浓度继续增大至300、400、500μmol／L时，表现出细胞毒作用，死亡细胞多于凋亡细胞，凋亡率非但没有继续增加，反而降低。2．就鸡胚尿囊绒毛膜血管生成而言，低浓度H₂O₂（1、2 nmol／cm²）轻微促进血管生成，4 nmol／cm² H₂O₂最大程度地促血管生成，而再高于此浓度则呈现抑制效果。可见，H₂O₂对于体内血管生成也具有双向调节作用。3．NADPH氧化酶抑制剂DPI对于正常状态和H₂O₂诱导的内皮细胞增殖、迁移、凋亡以及鸡胚尿囊绒毛膜血管生成均具有抑制作用，说明NADPH氧化酶介导了H₂O₂对血管生成的调节作用，同时也表明NADPH氧化酶是内皮细胞正常增殖、迁移以及体内血管生成过程中所需ROS的主要酶源。4．抗氧化剂马先蒿甙G降低了内皮细胞ROS的水平，抑制内皮细胞的增殖和迁移，以及在体血管生成。也降低了肝癌细胞内ROS的水平，抑制肝癌细胞的增殖和迁移，抑制接种于鸡胚尿囊绒毛膜的肝癌细胞形成肿瘤的生长。而预先经马先蒿甙G处理的肝癌细胞再接种于鸡胚尿囊绒毛膜上，使成瘤率减低，说明马先蒿甙G降低了癌细胞的毒性使癌细胞向正常逆转。总之，马先蒿甙G通过降低细胞内ROS水平，起到抗血管生成和抗肿瘤生成的双重作用。根据以上结果可以认为，以NADPH氧化酶介导产生的细胞活性氧，双相性地调控着体外及体内血管生成。当用抗氧化剂降低活性氧水平时，表现出抑制体内外血管生成，同时癌细胞的增殖也受到抑制，癌细胞发生了朝向正常化的逆转，即无论癌的发生和癌的生长都被抑制了。可见，NADPH氧化酶和抗氧化剂是血管生成、肿瘤发生的上游关键点和调控点。许多疾病如肿瘤、慢性炎症、糖尿病等的发生发展都依赖于血管生成，因此抑制血管生成有助于这些疾病的治疗；而对许多缺血性疾病如冠状动脉缺血、急性下肢缺血和脑梗塞等来说，促进血管生成可作为治疗的关键点之一。通过激活或抑制NADPH氧化酶以及选择抗氧化剂的剂量来调节细胞内活性氧的水平从而调控血管生成和肿瘤生成，可作为通过抗氧化来防治包括肿瘤或冠状动脉缺血等各种相关疾病的首选方案，本研究为此提供了基础数据。更多还原

【Abstract】 Angiogenesis is a complex physiological and pathological process that includes endothelial cell activation, extracellular matrix degradation, endothelial cell migration, proliferation and final new vessels formation. Reactive oxygen species （ROS） are believed to play an important role in the regulation of angiogenesis. ROS is the product of aerobic metabolism. A huge amount of ROS result in several kinds of deseases including cancer, whereas ROS in a moderate level have been regarded as signal molecules. The important signaling role of ROS during angiogenesis and tumorigenesis is thought highly and current studies are focused on the sources of ROS involved in angiogenesis and tumorigenesis. Although there are several ways to produce ROS in cells, multicomponent non-phagocytic NADPH oxidase seems to be especially important and be the one takes charge of the ROS generation in endothelial cells.Nicotinamide adenine dinucleotide phosphate oxidase （NADPH oxidase） mediated generation of reactive oxygen species （ROS） was originally identified as the powerful host defense machinery against microorganism in phagocytes. But after the mid-80s of last century, some reports indicated that some non-phagocytic cells, including endothelial cell, smooth muscle cell, and neurocyte also have the NADPH oxidase activity. It is reported that vascular NADPH oxidase is involved in atherosclerosis and diabetic vascular disorders.To clarify the role of ROS in angiogenesis and the potential mechanisms, the effects of extracellular H₂O₂, one kind of ROS, on endothelial cell proliferation, migration, apoptosis and angiogenesis in chicken embryo chorioallantoic membrane （CAM） as well as the role of NADPH oidase were studied. Meanwhile, A natural antioxidant, Pedicularioside G （Ped G）, on angiogenesis, tumorigenesis and formation of reactive oxygen species were studied.Results: 1. Low concentration of H₂O₂ （10μM） stimulates endothelial cell proliferation and migration, while higher concentration inhibits them. For apoptosis, low concentrations of H₂O₂ （under 100μM for 24 h） have almost no influence on apoptosis, while 200μM stimulated apoptosis strongly. However, much higher concentrations of H₂O₂, 300, 400 and 500μM, decline apoptosis with concentration dependently because of the toxicity of high level H₂O₂.2. H₂O₂, 1 or 2 nmol/cm², increases the neovascularization in chicken chorioallantoic membrane slightly, and get the peak at 4 nmol/cm², then declines with the concentration of H₂O₂ increasing.3. Endothelial cell proliferation, migration, apoptosis and neovascularization in chicken chorioallantoic membrane were significantly suppressed by addition of DPI, the inhibitor of NADPH oxidase, which means NADPH oxidase is implicated and may be activated by H₂O₂ to produce O₂ to carry out the angiogenesis processes.4. Ped G inhibits two prerequisite angiogenic procedures, endothelial cell proliferation and migration, as well as neovascularization in vivo. Moreover, Ped G inhibits human hepatoma cells proliferation and migration in vitro along with tumor formation and growth in vivo. Therefore, Ped G has the properties of not only anti-angiogenesis but also anti-tumor growth and anti-tumorigenesis which might partially attribute to its antioxidative activity.In conclusion, ROS mediated by NADPH oxidase regulated the angiogenesis in vitro and in vivo biphasically. Once the ROS in cells is reduced by antioxidants, both angiogenesis and tumorigenesis in vitro and in vivo will be suppressed. The results of the current study laid a foundation for the therapy of angiogenesis-releated diseases.更多还原