过氧化氢诱导的ECFC细胞氧化应激模型的氧化修饰亚蛋白质组学研究

发布时间：2018-07-13 15:36

【摘要】：内皮祖细胞（endothelialprogenitorcells,EPCs）是一类具有异质性的内皮前体细胞群体，已被证明在血管新生过程中有重要作用，并被视为一种评价血管疾病危险因素的生物学标志。研究表明，EPCs能够改善损伤内皮的功能并能延缓动脉粥样硬化的发展。血管内皮细胞的功能障碍与EPCs的功能损伤密切相关。氧化应激（oxidativestress）是导致内皮功能障碍最主要的病理因素之一。被氧化应激损伤的内皮细胞能够激活并释放一些细胞因子。这些细胞因子又能动员EPCs向损伤部位归巢并参与损伤内皮的修复。因此，参与内皮修复过程的EPCs势必暴露在氧化应激的环境中。大量研究证明高血压、高血脂、高血糖和动脉硬化等病理因素均可导致血管壁中活性氧（reactiveoxygenspecies，ROS）含量的增加。这些ROS所引起的促氧化反应又可造成氧化应激的出现。许多研究证实，心脑血管疾病患者体内的EPCs功能障碍、水平下降且与Framingham危险因素评分呈负相关。这说明氧化应激能够对EPCs造成损伤。但到目前为止，氧化应激对EPCs损伤的具体机制还不是十分清楚。EPCs可能和造血干细胞共同起源于中胚层卵黄囊的血岛。由于缺乏统一的鉴定和分离方法，所以在EPCs的鉴定和分离过程中有可能混入造血干细胞。造血干细胞可以分化成单核细胞和巨噬细胞。虽然以往有报道称EPCs有抗氧化能力，但其结果可能有局限性。因为其所分离的EPCs有可能混有具备抗氧化能力的巨噬细胞和单核细胞。为避免以往研究的不足之处，本实验以内皮克隆形成细胞（endothelial colony-forming cell,ECFC）作为研究对象。ECFC细胞是EPCs的一个亚群。但ECFC细胞不表达造血细胞、单核细胞或巨噬细胞的表面标志。因此，ECFC细胞被认为在分离过程中未混入造血干细胞。同时，ECFC细胞具有较高的增殖能力、表达相对高的端粒酶水平、并能在体内形成功能性血管。ECFC细胞在组织修复和再生方面的潜能也得到了一致的公认。为了深入探讨氧化应激对ECFC细胞氧化损伤的分子生物学机制，在蛋白质水平上寻找与ECFC细胞氧化损伤有关的氧化修饰的蛋白质，本研究首先应用过氧化氢（H_2O_2）处理ECFC细胞造成氧化应激的细胞模型。然后，通过MTT检测、AO/EB荧光染色和体外小管形成实验分析H_2O_2对ECFC细胞存活率、细胞凋亡和体外小管形成能力的影响。进一步，应用氧化亚蛋白质组学的研究方法，，分析氧化应激对ECFC细胞蛋白氧化水平的影响。经基质辅助激光解吸/电离-飞行时间串联质谱分析，鉴定氧化水平有显著差异的蛋白，揭示ECFC细胞氧化损伤现象背后的分子机制。结果显示，不同终浓度的H_2O_2（100μM、200μM、300μM）作用于ECFC细胞3小时后，与对照组相比较，实验组ECFC细胞的存活率随着H_2O_2浓度的增加而降低，分别为90.57±6.40%、85.06±6.00%和79.96±3.79%；实验组ECFC细胞的凋亡率随H_2O_2浓度的增加而升高，分别为8.67%、12.33%和20.00%；实验组完整小管个数和小管线性总长度均随着H_2O_2浓度的增加而减少，统计学上差异有显著性（P＜0.05）。氧化亚蛋白质组学分析发现有8个蛋白点氧化水平有显著差异。其中有5个蛋白点氧化水平上调，3个蛋白点氧化水平下调。经基质辅助激光解吸/电离-飞行时间串联质谱分析，有5个可信的鉴定结果，均为氧化水平上调的蛋白，氧化水平下调的3个蛋白点未被鉴定出来。这5个蛋白分别为T-complexprotein1subunitalpha、isoform A of prelamin-A/C、cofilin-1、peroxiredoxin-4和actin。它们在细胞运动、细胞凋亡、蛋白折叠和体外小管形成方面发挥着重要的作用。当这些蛋白发生氧化修饰后，其功能会有不同程度的受损。这些结果提示，在本实验的氧化应激细胞模型中，这5个蛋白的氧化修饰可能是ECFC细胞氧化损伤现象背后的分子机制之一。本研究的发现为探讨氧化应激对ECFC细胞的氧化损伤作用提供了新的线索，同时也为将ECFC细胞更好地应用于临床细胞治疗提供了新的理论依据。
[Abstract]:Endothelialprogenitorcells (EPCs), a group of heterogeneous endothelial progenitor cells, has been shown to play an important role in angiogenesis and is regarded as a biological marker for evaluating the risk factors of vascular disease. The study shows that EPCs can improve the function of damaged endothelium and delay atherosclerosis. The dysfunction of vascular endothelial cells is closely related to the functional damage of EPCs. Oxidative stress (oxidativestress) is one of the most important pathological factors that lead to endothelial dysfunction. Endothelial cells damaged by oxidative stress can activate and release some cytokines. These cell factors can also mobilize EPCs to return to the injured site and EPCs, which participates in the repair of damaged endothelium, therefore, is bound to be exposed to oxidative stress in the endothelium repair process. A large number of studies have proved that the pathological factors such as hypertension, hyperlipidemia, hyperglycemia, and arteriosclerosis can lead to an increase in the content of reactiveoxygenspecies (ROS) in the vascular wall. The oxidative reaction caused by these ROS It can cause oxidative stress. Many studies have confirmed that EPCs dysfunction in patients with cardiovascular and cerebrovascular diseases is negatively correlated with Framingham risk factors. This indicates that oxidative stress can cause damage to EPCs. But up to now, the specific mechanism of oxidative stress on EPCs damage is not very clear to.EPCs Hematopoietic stem cells originate from the blood island of the mesoderm yolk sac.
The separation method, therefore, may be mixed with hematopoietic stem cells during the identification and isolation of EPCs. Hematopoietic stem cells can differentiate into monocytes and macrophages. Although previously reported that EPCs has antioxidant capacity, the results may be limited, because the isolated EPCs can be mixed with macrophages with antioxidant capacity. In order to avoid the shortcomings of previous studies, the endothelial colony forming cells (endothelial) were used in this experiment.
Colony-forming cell, ECFC) as a study object,.ECFC cells are a subgroup of EPCs. But ECFC cells do not express the surface markers of hematopoietic cells, mononuclear cells or macrophages. Therefore, ECFC cells are considered to be not mixed with hematopoietic stem cells during the separation process. At the same time, ECFC cells have higher proliferation ability and express relatively high telomerase water. Flat, and can form functional vascular.ECFC in vivo, the potential of tissue repair and regeneration has been recognized.
In order to explore the molecular biological mechanism of oxidative stress on oxidative damage of ECFC cells and to search for the oxidative modified proteins associated with oxidative damage of ECFC cells at protein level, this study first used hydrogen peroxide (H_2O_2) to treat the oxidative stress caused by ECFC cells in a cell model. Then, MTT detection, AO/EB fluorescence staining was used. The effect of H_2O_2 on the survival rate of ECFC cells, cell apoptosis and the ability of tube formation in vitro. Further, the effect of oxidative stress on the oxidation level of ECFC cells was analyzed by the method of oxidative subprotein study. The analysis of the matrix assisted laser desorption / ionization time of flight tandem mass spectrometry was used to identify the effects of oxidative stress. The molecular mechanism behind the oxidative damage of ECFC cells was revealed by the significant difference in oxidation level. The results showed that the H_2O_2 (100, 200 M, 300 mu M) of different final concentrations acted on ECFC cells after 3 hours, and compared with the control group, the survival rate of ECFC cells in the experimental group decreased with the increase of H_2O_2 concentration, 90.57 + 6.40%, 85.06, respectively. The apoptosis rate of ECFC cells in the experimental group increased with the increase of H_2O_2 concentration, which were 8.67%, 12.33% and 20%, respectively. The total number of tubules and the linear total length of the tubules decreased with the increase of H_2O_2 concentration in the experimental group, and the statistical difference was significant (P < 0.05). The analysis of the oxidative subprotein group was 8. There were significant differences in the level of protein point oxidation. 5 protein spots were up up and 3 protein points were down down. Through matrix assisted laser desorption / ionization time of flight tandem mass spectrometry analysis, there were 5 credible results, all of which were up regulated by oxidation level, and 3 protein points of down regulation of oxidation level were not identified. These 5 points were not identified. Proteins are T-complexprotein1subunitalpha, isoform A of prelamin-A/C, cofilin-1, peroxiredoxin-4, and actin. play an important role in cell movement, cell apoptosis, protein folding and in vitro tubule formation. When these proteins are oxidized, their functions are impaired in varying degrees. These results suggest that In the oxidative stress cell model of this experiment, the oxidative modification of these 5 proteins may be one of the molecular mechanisms behind the oxidative damage of ECFC cells. The discovery of this study provides a new clue to explore the oxidative damage of ECFC cells by oxidative stress, and also provides a better application of ECFC cells to clinical cell therapy. A new theoretical basis.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R363

【共引文献】