【作者】 刘莹；

【导师】 胡林森；

【作者基本信息】 吉林大学 ， 神经病学， 2012， 博士

【摘要】 内皮祖细胞（endothelial progenitor cells, EPCs）可分化为内皮细胞，在组织损伤或者缺血时可被募集到损伤部位参与新生血管形成，以此来修复受损组织。目前的研究显示在有高脂血症、高血压、糖尿病、心脏病或者吸烟的脑血管病高危人群中，外周血EPCs数量降低、功能减退。它不仅是脑卒中的风险及预后预测指标，更因其能修复血管内皮、生成新生血管、延缓动脉粥样硬化斑块形成、促进神经发生而成为对于急性缺血性脑卒中患者具有治疗潜力的一类细胞。活性氧（reactive oxygen species, ROS）是需氧细胞在代谢过程中产生的，包括超氧阴离子（Ο2）、过氧化氢（H2O2）、超氧化氢离子（ΗΟ2）和羟自由基（ΟΗ）。许多病理条件例如高脂血症、高血压病、糖尿病会增加血管壁ROS的产生。另外，急性组织缺血也会导致微环境中ROS产生增多。目前的研究显示EPCs可通过高表达抗氧化酶类来对抗、防御氧化应激损伤，已被证实的抗氧化酶包括过氧化氢酶、锰超氧化物歧化酶（manganese superoxide dismutase，MnSOD）和谷胱甘肽过氧化物酶1（glutathione peroxidase-1，GPx-1）、血红素加氧酶1（haemoxygensae1，HO-1）。但是研究显示在同样的条件下,联合抑制EPCs前3种抗氧化酶后产生的ROS水平仍然较人脐静脉内皮细胞（human umbilicalvein endothelial cells，HUVECs）低,提示还有另外的抗氧化酶在EPCs中发挥清除ROS的作用。对于氧化损伤机制目前已知的有氧化应激使凋亡信号调控激酶1（apoptosis signal-regulating kinase1，ASK1）活性增高，导致细胞凋亡并由此引起血管形成能力降低。另外细胞内ROS产生的增多使人端粒酶逆转录酶（human telomerase reverse transcriptase，hTERT）活性降低，EPCs血管生成能力降低，同时增加的ROS刺激TERT从细胞核输出到细胞浆，导致端粒复制能力降低、延长能力丧失，端粒缩短，最终使细胞老化。其他有关氧化损伤机制的研究大多涉及高糖环境、氧化型低密度脂蛋白（oxidized low density lipoprotein，ox-LDL）、血管紧张素II（angiotensin II，Ang II）等因素导致的ROS升高，并且从这些因素本身对EPCs的影响来推测ROS对EPCs的作用，而对ROS与EPCs功能状态的直接作用机制了解甚少。本研究旨在通过蛋白质组学技术为EPCs的抗氧化机制和氧化损伤机制的研究寻找新的线索。本研究应用MTT比色法、划痕实验和小管形成实验对不同浓度（100、200、300和400μM）H2O2对EPCs作用3小时后的细胞数量、迁移能力和小管形成能力进行了比较，发现与对照组相比，细胞数量减少、迁移和小管形成能力下降，且这种负性作用为剂量依赖性。应用二维差异凝胶电泳（two-dimensional differential in-gelelectrophoresis，2D-DIGE）结合基质辅助激光解吸/电离-飞行时间质谱（matrix-assistedlaser desorption/ionization time of flight mass spectrometry, MALDI TOF/TOF MS）鉴定的方法对200μM H2O2处理3小时后的EPCs氧化应激模型进行了蛋白质组的分离和鉴定，发现了8个有明显表达差异的蛋白质，其中6个表达上调，2个表达下调。应用2D-Western blot方法选取其中一个蛋白进行验证，证明了质谱鉴定结果的正确性。EPCs通过高表达抗氧化蛋白过氧化还原酶2（peroxiredoxin-2，Prx-2）、硫氧还蛋白依赖性过氧化物还原酶即过氧化还原酶3（thioredoxin-dependent peroxide reductase/peroxiredoxin-3，Prx-3）、过氧化还原酶6（peroxiredoxin-6，Prx-6）和细胞骨架蛋白包含表皮生长因子腓骨蛋白样细胞外基质蛋白1（EGF-containing fibulin-like extracelluarmatrix protein1，EFEMP1）、波形蛋白（vimentin）来防御氧化应激损伤。对于氧化损伤的机制，我们的研究发现抑制信号转导的Rab GDP分离抑制剂α（Rab GDPdissociation inhibitor alpha，Rab GDI α）表达上调，参与清除氧化核苷酸、减少DNA复制错误的ADP糖焦磷酸酶（ADP-sugar pyrophosphatase，NUDT5）和催化“有氧糖酵解”产生能量的磷酸丙糖异构酶（triosephosphate isomerase，TIM）表达均下调。可见，尽管存在防御反应（包括抗氧化蛋白和细胞骨架蛋白的表达上调），由于与信号转导、氧化核苷酸清除、能量产生相关蛋白的表达改变，EPCs仍表现出数量减少、迁移和小管形成能力下降。这一发现提示我们今后在研究保护EPCs免受氧化应激损伤的关键点的工作中，不能仅仅关注于防御反应蛋白，更要重视像Rab GDIα、NUDT5和TIM这样的蛋白。因为这些负性通路不能被防御反应所保护，也许正是氧化应激造成EPCs功能紊乱的关键通路。本研究采用2D-DIGE结合MALDI TOF/TOF MS鉴定的方法研究EPCs氧化应激模型的蛋白质组学改变，这在国内外均未见报道，所鉴定的8个差异表达蛋白在EPCs氧化应激的研究中也均未见报道。抗氧化蛋白和细胞骨架蛋白表达上调的发现使EPCs抗氧化机制的研究得到进一步完善，而与信号转导、氧化核苷酸清除、能量产生相关蛋白的表达改变的发现为EPCs氧化损伤机制的研究提供了新的线索。更多还原

【Abstract】 Endothelial progenitor cells (EPCs) can differentiate into endothelial cells. When thetissue is damaged or ischemic, they can be recruited to the damaged area and participate inthe neovascularization to repair damaged tissue. The cumulative evidence indicates that inpatients with cerebrovascular risk factors, including hyperlipidemia, hypertension, diabetesmellitus, cardiovascular diseases, and also in smokers, there are reduced numbers andimpaired functionality of EPCs. They not only are the indicator of stroke risk and prognosis,but also have therapeutic potential to acute ischemic stroke patients because of the ability torepair vascular endothelium, form new vessels, delay the form of atherosclerosis plaque, andpromote neurogenesis.Reactive oxygen species (ROS) which includeΟ2, H2O2, ΗΟ2, ΟΗand so on areproduced in the process of metabolization by aerobic cells. Previous studies havedemonstrated that multiple pathological conditions can increase the production of ROS in thevascular wall, including hyperlipidemia, hypertension, and diabetes mellitus. In addition, theproduction of ROS is also increased in the environment of acute ischemia. It has been shownthat higher expression of intracellular antioxidative enzymes catalase, manganese superoxidedismutase (MnSOD), glutathione peroxidase-1(GPx-1), and haemoxygensae1(HO-1) arecritical mechanisms protecting EPCs against oxidative stress. But under the same condition,after inhibiting the first three antioxidative enzymes, the level of ROS in EPCs is still lowerthan that in human umbilical vein endothelial cells (HUVECs), indicating that there mustalso be some other antioxidative enzymes exist in EPCs to eliminate ROS. About themechanism of oxidative damage, one study has shown that enhanced apoptosissignal-regulating kinase1(ASK1) promoted apoptosis and leaded to the diminishedvessel-forming ability of EPCs after oxidative stress. Another study revealed that theincreased ROS impaired the telomerase reverse transcriptase (TERT) activity and inducedthe translocating of TERT protein from nucleus into the cytosol, finally reduced the prolongability of telomere which is followed by the onset of cell senescence. Other investigationsabout mechanisms of oxidative damage were indirect involving in different conditionsincluding high glucose condition, ox-LDL and angiotensin II, in which ROS production wasincreased. But the knowlege about oxidative stress and EPCs is limited. The aim of this study is to find new clues about antioxidative defensive mechanism and oxidative damagemechanism of EPCs with the help of proteomic analysis.To investigate the effect of oxidative stress on EPCs, cells were treated with H2O2atdifferent final concentrations (100、200、300and400μM) for3hours. MTT assay,scratch-wound assay and matrigel assay showed that cell number reduced, and migration andtubule formation function were impaired under H2O2stress in a concentration-dependentmanner. To identify proteins in response to H2O2stress, EPCs were treated in200μM H2O2for3hours and then two-dimensional differential in-gel electrophoresis (2D-DIGE) combinedwith matrix-assisted laser desorption/ionization time of flight mass spectrometry（MALDI-TOF/TOF MS) analysis were performed. Eight proteins were identifiedsuccessfully, six of which were up-regulated and the rest were down-regulated. To furtherconfirm the results from the MS analysis, the expression pattern of peroxiredoxin-3inresponse to H2O2stress was examined by2D-Western blot and the result showed that MS wasreliable. The results suggested that in order to protect themselves from oxidative damage,EPCs upregulated antioxidative enzymes peroxiredoxin-2, thioredoxin-dependent peroxidereductase, peroxiredoxin-6and cytoskeleton proteins EGF-containing fibulin-like extracelluarmatrix protein1and vimentin. About the oxidative damaged mechanism, we found that RabGDP dissociation inhibitor alpha which inhibited signal transduction was upregulated. BothADP-sugar pyrophosphatase which eliminated oxidative nucleotide and reduced errors duringDNA replication and triosephosphate isomerase which catalyzed “aerobic glycolysis” toproduce energy were downregulated. In summary, though the defensive responses existed,including the up-regulation of antioxidative enzymes and cytoskeleton proteins, EPCs stilldysfunctioned due to the altered proteins associated with signal transduction, oxidativenucleotide eliminating, and energy production. These results indicated that we should focuson the proteins which were susceptible to oxidative stress, as well as protective proteins, suchas Rab GDI α, NUDT5and TIM. Because these negative pathways can’t be avoided by theprotection of defensive responses and may be the key pathways of oxidative damage to EPCsfunctions.This study performed2D-DIGE combined with MALDI-TOF/TOF MS analysis to studythe proteomic alters of EPCs oxidative stress model. This hasn’t been reported in the literature.All of the eight altered proteins discovered in this study also haven’t been reported in theprevious studies of EPCs exposed to oxidative stress. The discovery of new antioxidativeenzymes and cytoskeleton proteins provide novel insights into antioxidative defensive mechanim of EPCs and the alteration of proteins associated with signal transduction,oxidative nucleotide eliminating, and energy production offers new clues to the study ofoxidative damage mechanism of EPCs.更多还原