過氧化氫誘導(dǎo)的ECFC細胞氧化應(yīng)激模型的氧化修飾亞蛋白質(zhì)組學研究
發(fā)布時間：2018-07-13 15:36
【摘要】：內(nèi)皮祖細胞（endothelialprogenitorcells,EPCs）是一類具有異質(zhì)性的內(nèi)皮前體細胞群體，已被證明在血管新生過程中有重要作用，并被視為一種評價血管疾病危險因素的生物學標志。研究表明，EPCs能夠改善損傷內(nèi)皮的功能并能延緩動脈粥樣硬化的發(fā)展。血管內(nèi)皮細胞的功能障礙與EPCs的功能損傷密切相關(guān)。氧化應(yīng)激（oxidativestress）是導(dǎo)致內(nèi)皮功能障礙最主要的病理因素之一。被氧化應(yīng)激損傷的內(nèi)皮細胞能夠激活并釋放一些細胞因子。這些細胞因子又能動員EPCs向損傷部位歸巢并參與損傷內(nèi)皮的修復(fù)。因此，參與內(nèi)皮修復(fù)過程的EPCs勢必暴露在氧化應(yīng)激的環(huán)境中。大量研究證明高血壓、高血脂、高血糖和動脈硬化等病理因素均可導(dǎo)致血管壁中活性氧（reactiveoxygenspecies，ROS）含量的增加。這些ROS所引起的促氧化反應(yīng)又可造成氧化應(yīng)激的出現(xiàn)。許多研究證實，心腦血管疾病患者體內(nèi)的EPCs功能障礙、水平下降且與Framingham危險因素評分呈負相關(guān)。這說明氧化應(yīng)激能夠?qū)PCs造成損傷。但到目前為止，氧化應(yīng)激對EPCs損傷的具體機制還不是十分清楚。EPCs可能和造血干細胞共同起源于中胚層卵黃囊的血島。由于缺乏統(tǒng)一的鑒定和 分離方法，所以在EPCs的鑒定和分離過程中有可能混入造血干細胞。造血干細胞可以分化成單核細胞和巨噬細胞。雖然以往有報道稱EPCs有抗氧化能力，但其結(jié)果可能有局限性。因為其所分離的EPCs有可能混有具備抗氧化能力的巨噬細胞和單核細胞。為避免以往研究的不足之處，本實驗以內(nèi)皮克隆形成細胞（endothelial colony-forming cell,ECFC）作為研究對象。ECFC細胞是EPCs的一個亞群。但ECFC細胞不表達造血細胞、單核細胞或巨噬細胞的表面標志。因此，ECFC細胞被認為在分離過程中未混入造血干細胞。同時，ECFC細胞具有較高的增殖能力、表達相對高的端粒酶水平、并能在體內(nèi)形成功能性血管。ECFC細胞在組織修復(fù)和再生方面的潛能也得到了一致的公認。 為了深入探討氧化應(yīng)激對ECFC細胞氧化損傷的分子生物學機制，在蛋白質(zhì)水平上尋找與ECFC細胞氧化損傷有關(guān)的氧化修飾的蛋白質(zhì)，本研究首先應(yīng)用過氧化氫（H_2O_2）處理ECFC細胞造成氧化應(yīng)激的細胞模型。然后，通過MTT檢測、AO/EB熒光染色和體外小管形成實驗分析H_2O_2對ECFC細胞存活率、細胞凋亡和體外小管形成能力的影響。進一步，應(yīng)用氧化亞蛋白質(zhì)組學的研究方法，，分析氧化應(yīng)激對ECFC細胞蛋白氧化水平的影響。經(jīng)基質(zhì)輔助激光解吸/電離-飛行時間串聯(lián)質(zhì)譜分析，鑒定氧化水平有顯著差異的蛋白，揭示ECFC細胞氧化損傷現(xiàn)象背后的分子機制。結(jié)果顯示，不同終濃度的H_2O_2（100μM、200μM、300μM）作用于ECFC細胞3小時后，與對照組相比較，實驗組ECFC細胞的存活率隨著H_2O_2濃度的增加而降低，分別為90.57±6.40%、85.06±6.00%和79.96±3.79%；實驗組ECFC細胞的凋亡率隨H_2O_2濃度的增加而升高，分別為8.67%、12.33%和20.00%；實驗組完整小管個數(shù)和小管線性總長度均隨著H_2O_2濃度的增加而減少，統(tǒng)計學上差異有顯著性（P＜0.05）。氧化亞蛋白質(zhì)組學分析發(fā)現(xiàn)有8個蛋白點氧化水平有顯著差異。其中有5個蛋白點氧化水平上調(diào)，3個蛋白點氧化水平下調(diào)。經(jīng)基質(zhì)輔助激光解吸/電離-飛行時間串聯(lián)質(zhì)譜分析，有5個可信的鑒定結(jié)果，均為氧化水平上調(diào)的蛋白，氧化水平下調(diào)的3個蛋白點未被鑒定出來。這5個蛋白分別為T-complexprotein1subunitalpha、isoform A of prelamin-A/C、cofilin-1、peroxiredoxin-4和actin。它們在細胞運動、細胞凋亡、蛋白折疊和體外小管形成方面發(fā)揮著重要的作用。當這些蛋白發(fā)生氧化修飾后，其功能會有不同程度的受損。這些結(jié)果提示，在本實驗的氧化應(yīng)激細胞模型中，這5個蛋白的氧化修飾可能是ECFC細胞氧化損傷現(xiàn)象背后的分子機制之一。本研究的發(fā)現(xiàn)為探討氧化應(yīng)激對ECFC細胞的氧化損傷作用提供了新的線索，同時也為將ECFC細胞更好地應(yīng)用于臨床細胞治療提供了新的理論依據(jù)。
[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.
【學位授予單位】：吉林大學
【學位級別】：博士
【學位授予年份】：2012
【分類號】：R363
本文編號：2119905
[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.
【學位授予單位】：吉林大學
【學位級別】：博士
【學位授予年份】：2012
【分類號】：R363
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