S-亚硝基谷胱甘肽经Peroxiredoxin-2亚硝基化促小鼠胚胎干细胞心肌分化机制研究

发布时间：2019-06-14 19:14

【摘要】：胚胎干(Embryonic Stem, ES)细胞是全能性干细胞的典型代表,其心肌分化过程可以用来研究心肌发育过程,而其分化而来的心肌细胞可以作为细胞治疗的供体或作为药物筛选平台。近年来,一氧化氮(Nitric Oxide, NO)作为一种气体信号分子,成为基础生物学、生物医学及转化医学领域的明星分子,在逐步揭开其生理作用的同时,围绕其进行的临床研究也取得了丰硕的成果,开发出了多种药物和治疗方法,为人类健康做出了重要贡献。由于在几乎所有人体组织中都有NO合酶(NOS)的表达,而且NO在干细胞增殖分化、表观遗传学调控和免疫反应等方面都发挥重要作用,所以NO必然和干细胞基础研究及临床应用有着密切的相关性。本研究论证了NO供体S-亚硝基谷胱甘肽(S-Nitrosoglutathione, GSNO)短时间处理可以通过亚硝基化作用促进心肌分化。亚硝基化修饰是指NO基团与蛋白质巯基,尤其是半胱氨酸上活性巯基的结合,可以发挥多种生物学功能,如调节蛋白质稳定性、反应活性,干扰蛋白质间相互结合以及细胞内定位,甚至与其他种类翻译后修饰竞争等。GSNO作为体内广泛存在的亚硝基硫醇类NO供体,是NO在体内的主要承载体,也是细胞内蛋白质亚硝基化修饰功能的主要承担者。在心脏中,GSNO是一种内源性保护性物质并且可以对多种蛋白进行亚硝基化修饰,对于心肌组织中多种离子通道、酶类发挥正常功能,维持心肌细胞内氧还平衡等至关重要。那么,研究GSNO在干细胞心肌发生中的作用有着非常重要的价值,将有可能对再生医学和心肌修复领域提供有意义的参考。至今为止,绝大多数关于NO在干细胞生物学方面的研究集中于sGC/cGMP途径,而忽视了对蛋白亚硝基化可能发挥的作用。亚硝基化修饰靶点多样,功能各异,在干细胞心肌分化过程中可以发挥何种作用值得探究。在本研究中,发现短时间GSNO处理ES细胞衍生的拟胚体(Embryoid Body,EB)可以提高心肌细胞分化率,并阐明亚硝基化在这一过程中发挥的关键作用。通过定量亚硝基化蛋白组学和生物信息学分析,发现Prdx-2的亚硝基化可以导致过氧化氢(H202)积聚并启动X-box binding protein-1s/PI3K/AKT信号通路,最终促进了心肌细胞的分化。这些发现对干细胞分化的调控提供了新的认识,并对再生医学和诱导多能干(iPS)细胞治疗提供了有益的借鉴。一.S-亚硝基谷胱甘肽促胚胎干细胞心肌分化及亚硝基化蛋白组学目的：探索GSNO对小鼠ES细胞心肌分化的影响,同时论证GSNO起作用的方式。方法和结果：采用悬滴、悬浮、贴壁三步法诱导心肌分化体系,在形成EBs后,对EBs进行2h的GSNO处理,然后继续贴壁培养并考察心肌分化情况。结果发现,25μM GSNO处理可以显著提高贴壁培养3d后EBs搏动率和心肌特异蛋白a-actinin表达,免疫荧光也显示GSNO可以诱导形成结构完整的肌小节结构。通过亚硝基化还原剂DTT、sGC抑制剂ODQ和GSNO合用,发现DTT可以拮抗GSNO的促心肌分化作用,而ODQ不能拮抗,说明GSNO的促分化作用是依赖于其亚硝基化作用。继而采用氨基酸稳定同位素标记(Stable Isotope Labeling with Amino Acidsin Cell Cultures, SILAC)法对细胞进行同位素标记,然后在GSNO处理后提取亚硝基化蛋白,通过质谱检测GSNO处理导致亚硝基化蛋白靶点并定量分析亚硝基化程度,通过生物信息学分析对亚硝基化蛋白进行分类。GSNO处理后,发现104个亚硝基化上调蛋白,其中既具有酶活性又与氧还平衡密切相关的Prdx家族1和2型蛋白亚硝基化分别上升2.66和3.06倍,是被亚硝基化程度较高的蛋白。结论：GSNO可通过蛋白质亚硝基化作用促进心肌分化,明确了104个蛋白亚硝基化靶点,通过蛋白功能分类,选择亚硝基化修饰程度高,同时既具有酶活性又与氧还平衡密切相关的Prdx-1,2为后续论证的候选靶蛋白。二. Peroxiredoxin-2亚硝基化促进心肌分化机制研究目的：探索Prdx-2亚硝基化在GSNO促心肌分化中的作用方法和结果：首先对质谱结果进行免疫印迹确认,发现相对于Prdx-1, Prdx-2是拟胚体时期高表达的Prdx类型；继而考察GSNO处理后,Prdx-2底物过氧化氢(H202)含量变化情况,发现GSNO处理后,EB内H202积聚,而前体超氧阴离子(02·-)并没有增加。同时发现p38 MAPK并没有被GSNO激活,而PI3K/Akt信号通路被激活。通过对P13K不同亚基的检测,发现p-p85 PI3K亚基在GSNO处理后,在胞浆胞核的含量发生变化,更多地聚集在胞浆中,同时与p110 PI3K亚基结合上升,激活P13K通路。对p85 PI3K亚基入核分子伴侣XBP-1s检测发现GSNO和H202处理均不影响其表达和细胞内定位,然而对其和p-p85 PI3K亚基的结合有下调作用。干扰XBP-1s表达可以激活PI3K/Akt信号通路,并促进ES细胞心肌分化。结论：GSNO致Prdx-2亚硝基化可抑制Prdx-2催化H_2O_2降解,使细胞内特定区域H_2O_2积聚,通过减弱)CBP-1s 和 p-p85 PI3K亚基的结合,激活P13K/Akt信号通路,促进ES细胞心肌分化。
[Abstract]:Embryonic stem (ES) cells are representative of functional stem cells, and their cardiac differentiation processes can be used to study the development of the myocardium, and the differentiated cardiomyocytes can be used as a donor for cell therapy or as a drug screening platform. In recent year, nitric oxide (NO), as a gas signal molecule, is a star molecule in that field of basic biology, biomedicine and transformation. A variety of drugs and methods of treatment have been developed to make an important contribution to human health. NO synthase (NOS) is expressed in almost all human tissues, and it plays an important role in stem cell proliferation, differentiation, epigenetic regulation and immune response, so there is a close correlation between NO and stem cell basic research and clinical application. This study demonstrated that the short-time treatment of NO donor S-nitrosoglutathione (GSNO) could promote the differentiation of cardiac muscle through the action of nitroso. The nitroso-based modification refers to the combination of the NO group and the active carbon-based group of the protein, in particular cysteine, and can play a plurality of biological functions, such as regulating the stability of the protein, the activity of the reaction, the mutual combination of the interfering proteins and the intracellular positioning, Or even post-translational modification of competition, and the like. GSNO, which is the main carrier of NO in the body, is the main carrier of the nitroso-type NO donor in the body, and is the main undertaker of the nitroso-modified function of the protein in the cell. In the heart, GSNO is an endogenous protective substance and can be modified by nitroso on a plurality of proteins, which is of vital importance to various ion channels in the myocardial tissue, the normal function of the enzymes, the maintenance of oxygen in the cardiac muscle cells, and the like. So, it is of great value to study the role of GSNO in the development of stem cells, and it is possible to provide meaningful reference to the field of regenerative medicine and myocardial repair. Up to now, the vast majority of the studies on the biological aspects of stem cells have focused on the sGC/ cGMP pathway, while ignoring the potential role for protein nitroso. The role of nitroso-modified target in the differentiation of stem cells can be explored. In this study, it was found that a short-time GNO-treated ES cell-derived embryoid body (EB) could increase the rate of differentiation of the cardiac muscle cells and clarify the key role of nitroso in this process. It was found that the nitrosomatization of Prdx-2 could lead to the accumulation of hydrogen peroxide (H202) and the initiation of the X-box binding protein-1s/ PI3K/ AKT signal pathway by quantitative nitroso proteomics and bioinformatics. These findings provide a new understanding of the regulation of stem cell differentiation and provide a useful reference for regenerative medicine and the induction of multipotent (iPS) cell therapy. I. Objective: To explore the effect of GSNO on myocardial differentiation of mouse ES cells and to demonstrate the effect of GSNO on the differentiation of mouse ES cells. Methods and Results: The myocardial differentiation system was induced by three-step method of hanging drop, suspension and malapposition. After the formation of the EBs, the EBs were treated with GSNO for 2 hours, then the adherent cells were continued to be cultured and the differentiation of the myocardium was examined. The results showed that 25. mu. M GSNO treatment could significantly increase the rate of EBs and the expression of the specific protein a-actin after 3 days of the adherent culture, and the immunofluorescence also showed that the GSNO could induce the formation of a complete muscle section structure. DTT, sGC inhibitor ODQ and GSNO were used in combination with the nitroso-reducing agent DTT, the sGC inhibitor ODQ and the GSNO, and it was found that the DTT could antagonize the growth-promoting differentiation of GSNO, and the ODQ could not antagonize. The cell is labeled with an amino acid stable isotope labeling (SILAC) method, and then the nitroso protein is extracted after the GSNO treatment, and the nitroso protein target is caused by the detection of the GSNO treatment by mass spectrometry, and the degree of the nitroso is quantitatively analyzed, The nitroso-treated protein was classified by bioinformatics analysis. After the treatment of GSNO,104 nitrosolated up-regulated proteins were found, in which the Prdx family 1 and 2-type protein, which had both enzymatic activity and oxygen balance, increased by 2.66 and 3.06 times, respectively, and was a higher degree of nitroso-treated protein. Conclusion: GSNO can promote the differentiation of the myocardium by the action of the nitroso-nitrosation of the protein, define the target of the nitroso-nitrosation of 104 proteins, and select the Prdx-1,2, which is closely related to the balance of the oxygen and the enzyme activity, as well as the candidate target protein which is closely related to the oxygen balance. II. Objective: To study the effect of Prdx-2 nitroso on the differentiation of the cardiac muscle of GSNO: to study the effect of Prdx-2 nitroso on the differentiation of the cardiac muscle of GSNO: first, immunoblotting the results of the mass spectra, and finding that Prdx-1 and Prdx-2 are the high-expressed Prdx types in the time of the quasi-embryonic body; and then, after the GNO treatment, The content of hydrogen peroxide (H202) in Prdx-2 substrate was changed, and after the treatment of GSNO, the accumulation of H202 in EB was found, and the precursor superoxide anion (02.-) did not increase. At the same time, it was found that p38 MAPK was not activated by GSNO and the PI3K/ Akt signal pathway was activated. Through the detection of the different subunits of P13K, it was found that the content of p-p85 PI3K in the cytoplasm of the cytoplasm of the cytoplasm of the p13K was changed, and it was more concentrated in the cytoplasm, and the P13K pathway was activated in combination with the p110 PI3K subunit. The detection of p85 PI3K subunit in the nuclear molecular chaperone XBP-1s showed that both GSNO and H202 treatment did not affect its expression and intracellular localization, however, the binding of the p85 PI3K subunit to the p-p85 PI3K subunit was down-regulated. The expression of the interfering XBP-1s can activate the PI3K/ Akt signaling pathway and promote the differentiation of the ES cells. Conclusion: The P13K/ Akt signal pathway is activated by the combination of the CBP-1s and the p-p85 PI3K subunits, and the differentiation of the ES cells is promoted by the combination of the CBP-1s and the p-p85 PI3K subunits.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R96

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