發(fā)布時間：2019-06-14 19:14

【摘要】：胚胎干(Embryonic Stem, ES)細(xì)胞是全能性干細(xì)胞的典型代表,其心肌分化過程可以用來研究心肌發(fā)育過程,而其分化而來的心肌細(xì)胞可以作為細(xì)胞治療的供體或作為藥物篩選平臺。近年來,一氧化氮(Nitric Oxide, NO)作為一種氣體信號分子,成為基礎(chǔ)生物學(xué)、生物醫(yī)學(xué)及轉(zhuǎn)化醫(yī)學(xué)領(lǐng)域的明星分子,在逐步揭開其生理作用的同時,圍繞其進行的臨床研究也取得了豐碩的成果,開發(fā)出了多種藥物和治療方法,為人類健康做出了重要貢獻。由于在幾乎所有人體組織中都有NO合酶(NOS)的表達,而且NO在干細(xì)胞增殖分化、表觀遺傳學(xué)調(diào)控和免疫反應(yīng)等方面都發(fā)揮重要作用,所以NO必然和干細(xì)胞基礎(chǔ)研究及臨床應(yīng)用有著密切的相關(guān)性。本研究論證了NO供體S-亞硝基谷胱甘肽(S-Nitrosoglutathione, GSNO)短時間處理可以通過亞硝基化作用促進心肌分化。亞硝基化修飾是指NO基團與蛋白質(zhì)巰基,尤其是半胱氨酸上活性巰基的結(jié)合,可以發(fā)揮多種生物學(xué)功能,如調(diào)節(jié)蛋白質(zhì)穩(wěn)定性、反應(yīng)活性,干擾蛋白質(zhì)間相互結(jié)合以及細(xì)胞內(nèi)定位,甚至與其他種類翻譯后修飾競爭等。GSNO作為體內(nèi)廣泛存在的亞硝基硫醇類NO供體,是NO在體內(nèi)的主要承載體,也是細(xì)胞內(nèi)蛋白質(zhì)亞硝基化修飾功能的主要承擔(dān)者。在心臟中,GSNO是一種內(nèi)源性保護性物質(zhì)并且可以對多種蛋白進行亞硝基化修飾,對于心肌組織中多種離子通道、酶類發(fā)揮正常功能,維持心肌細(xì)胞內(nèi)氧還平衡等至關(guān)重要。那么,研究GSNO在干細(xì)胞心肌發(fā)生中的作用有著非常重要的價值,將有可能對再生醫(yī)學(xué)和心肌修復(fù)領(lǐng)域提供有意義的參考。至今為止,絕大多數(shù)關(guān)于NO在干細(xì)胞生物學(xué)方面的研究集中于sGC/cGMP途徑,而忽視了對蛋白亞硝基化可能發(fā)揮的作用。亞硝基化修飾靶點多樣,功能各異,在干細(xì)胞心肌分化過程中可以發(fā)揮何種作用值得探究。在本研究中,發(fā)現(xiàn)短時間GSNO處理ES細(xì)胞衍生的擬胚體(Embryoid Body,EB)可以提高心肌細(xì)胞分化率,并闡明亞硝基化在這一過程中發(fā)揮的關(guān)鍵作用。通過定量亞硝基化蛋白組學(xué)和生物信息學(xué)分析,發(fā)現(xiàn)Prdx-2的亞硝基化可以導(dǎo)致過氧化氫(H202)積聚并啟動X-box binding protein-1s/PI3K/AKT信號通路,最終促進了心肌細(xì)胞的分化。這些發(fā)現(xiàn)對干細(xì)胞分化的調(diào)控提供了新的認(rèn)識,并對再生醫(yī)學(xué)和誘導(dǎo)多能干(iPS)細(xì)胞治療提供了有益的借鑒。一.S-亞硝基谷胱甘肽促胚胎干細(xì)胞心肌分化及亞硝基化蛋白組學(xué)目的：探索GSNO對小鼠ES細(xì)胞心肌分化的影響,同時論證GSNO起作用的方式。方法和結(jié)果：采用懸滴、懸浮、貼壁三步法誘導(dǎo)心肌分化體系,在形成EBs后,對EBs進行2h的GSNO處理,然后繼續(xù)貼壁培養(yǎng)并考察心肌分化情況。結(jié)果發(fā)現(xiàn),25μM GSNO處理可以顯著提高貼壁培養(yǎng)3d后EBs搏動率和心肌特異蛋白a-actinin表達,免疫熒光也顯示GSNO可以誘導(dǎo)形成結(jié)構(gòu)完整的肌小節(jié)結(jié)構(gòu)。通過亞硝基化還原劑DTT、sGC抑制劑ODQ和GSNO合用,發(fā)現(xiàn)DTT可以拮抗GSNO的促心肌分化作用,而ODQ不能拮抗,說明GSNO的促分化作用是依賴于其亞硝基化作用。繼而采用氨基酸穩(wěn)定同位素標(biāo)記(Stable Isotope Labeling with Amino Acidsin Cell Cultures, SILAC)法對細(xì)胞進行同位素標(biāo)記,然后在GSNO處理后提取亞硝基化蛋白,通過質(zhì)譜檢測GSNO處理導(dǎo)致亞硝基化蛋白靶點并定量分析亞硝基化程度,通過生物信息學(xué)分析對亞硝基化蛋白進行分類。GSNO處理后,發(fā)現(xiàn)104個亞硝基化上調(diào)蛋白,其中既具有酶活性又與氧還平衡密切相關(guān)的Prdx家族1和2型蛋白亞硝基化分別上升2.66和3.06倍,是被亞硝基化程度較高的蛋白。結(jié)論：GSNO可通過蛋白質(zhì)亞硝基化作用促進心肌分化,明確了104個蛋白亞硝基化靶點,通過蛋白功能分類,選擇亞硝基化修飾程度高,同時既具有酶活性又與氧還平衡密切相關(guān)的Prdx-1,2為后續(xù)論證的候選靶蛋白。二. Peroxiredoxin-2亞硝基化促進心肌分化機制研究目的：探索Prdx-2亞硝基化在GSNO促心肌分化中的作用方法和結(jié)果：首先對質(zhì)譜結(jié)果進行免疫印跡確認(rèn),發(fā)現(xiàn)相對于Prdx-1, Prdx-2是擬胚體時期高表達的Prdx類型；繼而考察GSNO處理后,Prdx-2底物過氧化氫(H202)含量變化情況,發(fā)現(xiàn)GSNO處理后,EB內(nèi)H202積聚,而前體超氧陰離子(02·-)并沒有增加。同時發(fā)現(xiàn)p38 MAPK并沒有被GSNO激活,而PI3K/Akt信號通路被激活。通過對P13K不同亞基的檢測,發(fā)現(xiàn)p-p85 PI3K亞基在GSNO處理后,在胞漿胞核的含量發(fā)生變化,更多地聚集在胞漿中,同時與p110 PI3K亞基結(jié)合上升,激活P13K通路。對p85 PI3K亞基入核分子伴侶XBP-1s檢測發(fā)現(xiàn)GSNO和H202處理均不影響其表達和細(xì)胞內(nèi)定位,然而對其和p-p85 PI3K亞基的結(jié)合有下調(diào)作用。干擾XBP-1s表達可以激活PI3K/Akt信號通路,并促進ES細(xì)胞心肌分化。結(jié)論：GSNO致Prdx-2亞硝基化可抑制Prdx-2催化H_2O_2降解,使細(xì)胞內(nèi)特定區(qū)域H_2O_2積聚,通過減弱)CBP-1s 和 p-p85 PI3K亞基的結(jié)合,激活P13K/Akt信號通路,促進ES細(xì)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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R96

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6 孔慶南;桃果實蛋白質(zhì)S-亞硝基化作用及SPR檢測亞硝基硫醇方法的研究[D];山東農(nóng)業(yè)大學(xué);2013年

7 邱蔥花;水楊酸酯與硝酸釔配位誘導(dǎo)下氮氧化合物的形成與亞硝基化反應(yīng)[D];南昌大學(xué);2007年

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