本文選題：絲/蘇氨酸磷酸酶2A催化亞單位C(PP2Ac) + 酪氨酸位點(diǎn)硝基化�。� 參考：《華中科技大學(xué)》2016年博士論文

【摘要】：目的腎小管管周毛細(xì)血管內(nèi)皮細(xì)胞向間充質(zhì)轉(zhuǎn)分化(EndMT)是新近發(fā)現(xiàn)的腎間質(zhì)肌成纖維細(xì)胞的重要來(lái)源,其發(fā)生機(jī)制與內(nèi)皮細(xì)胞緊密連接失去功能密切相關(guān)。研究表明蛋白絲/蘇氨酸磷酸酶2A (PP2A)的催化亞單位C (PP2Ac)酪氨酸位點(diǎn)硝基化可激活PP2A并能下調(diào)緊密連接連接蛋白(Occludin)絲/蘇氨酸位點(diǎn)磷酸化,從而參與內(nèi)皮細(xì)胞緊密連接的功能調(diào)控。本研究探討PP2Ac酪氨酸核心位點(diǎn)硝基化對(duì)內(nèi)皮細(xì)胞緊密連接的影響,并闡明該位點(diǎn)是腎小管管周毛細(xì)血管EndMT的重要調(diào)控分子,參與進(jìn)行性腎間質(zhì)纖維化,為探索防治腎間質(zhì)纖維化進(jìn)行性發(fā)展的新治療靶點(diǎn)提供實(shí)驗(yàn)依據(jù)。方法體外構(gòu)建硝基化反應(yīng)體系,用過(guò)氧化亞硝酸鹽(Peroxynitrite)刺激重組的PP2Ac蛋白,western blotting收集對(duì)照組和刺激組中的蛋白行質(zhì)譜檢測(cè),分析PP2Ac酪氨酸發(fā)生硝基化的具體位點(diǎn)。對(duì)上述硝基化位點(diǎn)進(jìn)行計(jì)算機(jī)模擬分析以明確各位點(diǎn)空間暴露程度從而確定調(diào)控PP2A活性的核心位點(diǎn)。同時(shí)構(gòu)建、合成以核心位點(diǎn)為中心的底物模擬多肽和對(duì)照多肽,體外實(shí)驗(yàn)中驗(yàn)證該多肽對(duì)EndMT的干預(yù)作用,體內(nèi)實(shí)驗(yàn)首先應(yīng)用活體成像技術(shù)檢測(cè)多肽在小鼠體內(nèi)的分布以及代謝狀態(tài),然后通過(guò)尾靜脈聯(lián)合腹腔多肽注射觀察該多肽在UUO小鼠模型中對(duì)管周毛細(xì)血管EndMT的干預(yù)效應(yīng)。結(jié)果：質(zhì)譜檢測(cè)分析PP2Ac發(fā)生硝基化的具體位點(diǎn)有Y284/267/265/218/130/127 。在這六個(gè)位點(diǎn)中,Y127在蛋白質(zhì)空間結(jié)構(gòu)、相鄰氨基酸電荷分布上具有更穩(wěn)定的空間表位優(yōu)勢(shì),在PP2Ac活化過(guò)程中起核心作用。以127位點(diǎn)為中心構(gòu)建及合成的可穿透性底物模擬多肽和對(duì)照多肽能夠抑制TGF-β1誘導(dǎo)的EndMT。體外試驗(yàn)中,多肽在肝臟和腎臟中具有較高的藥物積聚；較對(duì)照多肽,底物模擬多肽可明顯抑制Occludin去磷酸化,減少EndMT發(fā)生,穩(wěn)定外周毛細(xì)血管結(jié)構(gòu)并部分減輕腎間質(zhì)纖維化。結(jié)論以PP2Ac Y127為核心的底物模擬多肽可有效干預(yù)內(nèi)皮細(xì)胞轉(zhuǎn)分化,是腎間質(zhì)纖維化治療的潛在生物靶點(diǎn)。第一部分管周毛細(xì)血管EndMT是腎間質(zhì)纖維化的重要組成部分目的在原發(fā)和繼發(fā)性腎臟疾病以及UUO動(dòng)物模型中探討EndMT發(fā)生的證據(jù),觀察轉(zhuǎn)化生長(zhǎng)因子β1 (Transforming growth factor beta 1, TGF-β1)促進(jìn)內(nèi)皮細(xì)胞間充質(zhì)轉(zhuǎn)分化效應(yīng),探討腎間質(zhì)纖維化肌成纖維細(xì)胞內(nèi)皮源性。方法選取原發(fā)性腎臟疾病(IgA腎病)以及繼發(fā)性腎臟疾病(狼瘡腎炎和糖尿病腎病)腎活檢標(biāo)本,應(yīng)用血管內(nèi)皮細(xì)胞特異性標(biāo)志物CD31和肌成纖維細(xì)胞特異性標(biāo)志物a-SMA行連續(xù)切片免疫熒光雙標(biāo)檢測(cè),并在陽(yáng)性區(qū)域進(jìn)行共聚焦三維結(jié)構(gòu)重建明確CD31+a-SMA+共表達(dá)。體內(nèi)實(shí)驗(yàn)采用C57小鼠單側(cè)輸尿管梗阻腎病模型,熒光雙標(biāo)結(jié)合三維重建觀察CD31+a-SMA+共表達(dá)情況。體外實(shí)驗(yàn)采用TGF-β1 (lOng/ml)刺激人臍靜脈內(nèi)皮細(xì)胞72h建立EndMT細(xì)胞模型,鏡下觀察細(xì)胞形態(tài)變化,免疫熒光及western blot檢測(cè)血管內(nèi)皮鈣粘素(VE-cadherin,內(nèi)皮特異性標(biāo)志物)和平滑肌肌動(dòng)蛋白(a-SMA)表達(dá)變化。結(jié)果和對(duì)照組相比,在IgA腎病、狼瘡腎炎、糖尿病腎病以及UUO動(dòng)物模型腎間質(zhì)纖維化區(qū)域,管周毛細(xì)血管管腔異常膨大或縮小;共聚焦顯微鏡下觀察發(fā)現(xiàn)CD31和α-SMA共表達(dá)增多,差異具有統(tǒng)計(jì)學(xué)意義(P0.05)；對(duì)該共表達(dá)部位血管進(jìn)行三維立體重建,發(fā)現(xiàn)部分內(nèi)皮細(xì)胞可表達(dá)α-SMA。體外實(shí)驗(yàn),TGF-β1刺激72h后,內(nèi)皮細(xì)胞形態(tài)由鋪路石狀向梭型轉(zhuǎn)化,細(xì)胞與細(xì)胞間距明顯增大;和對(duì)照組相比,肌成纖維細(xì)胞標(biāo)志物α-SMA表達(dá)顯著增加,而內(nèi)皮細(xì)胞標(biāo)志物VE-cadherin表達(dá)下調(diào),差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。結(jié)論在原發(fā)和繼發(fā)性腎臟病中,內(nèi)皮細(xì)胞間充質(zhì)轉(zhuǎn)分化是腎間質(zhì)纖維化肌成纖維細(xì)胞的重要組成部分。內(nèi)皮細(xì)胞受到促纖維化因子刺激后,細(xì)胞表型和功能特性均可向間充質(zhì)細(xì)胞轉(zhuǎn)化。第二部分PP2A活化及PP2Ac硝基化對(duì)EndMT的作用研究目的探討內(nèi)皮細(xì)胞PP2A活化及PP2Ac硝基化對(duì)EndMT進(jìn)程的影響方法(1)提取UUO小鼠不同梗阻時(shí)間點(diǎn)下腎臟組織蛋白以及不同TGF-β1刺激時(shí)間點(diǎn)下細(xì)胞總蛋白,繼而用絲／蘇氨酸蛋白磷酸酶活性試劑盒監(jiān)測(cè)PP2A的活性變化;(2)采用PP2A特異性抑制劑岡田酸(Okadaic acid, OA)預(yù)處理后,觀察TGF-β1刺激下α-SMA、VE-cadherin蛋白表達(dá)變化以及PP2A底物緊密連接蛋白Occludin磷酸化水平;(3) PP2Ac是PP2A的活性亞基,運(yùn)用免疫組化及免疫熒光定位PP2Ac在腎臟組織的表達(dá),并通過(guò)免疫共沉淀檢測(cè)TGF-β1刺激下內(nèi)皮細(xì)胞PP2Ac翻譯后修飾變化及其對(duì)PP2A磷酸酶活性的影響。結(jié)果(1)和對(duì)照組相比,隨著梗阻時(shí)間延長(zhǎng),PP2A活性逐漸增加并在UUO-14d達(dá)到峰值;體外實(shí)驗(yàn),TGF-β1刺激下,PP2A的活性在15min開(kāi)始升高,在60min時(shí)顯著增強(qiáng)(P0.05) 。 (2) western blot結(jié)果顯示較TGF-β1組,OA干預(yù)組α-SMA表達(dá)顯著下調(diào)而VE-cadherin表達(dá)顯著上調(diào)；免疫共沉淀示正常HUVE細(xì)胞緊密連接蛋白Occludin磷酸化水平維持在較高水平,TGF-β1刺激72h后磷酸化水平明顯降低,OA預(yù)處理后可部分抑制Occludin磷酸化減少(P0.05)。(3)免疫熒光檢測(cè)可見(jiàn)PP2Ac主要在腎小球及腎間質(zhì)毛細(xì)血管內(nèi)皮細(xì)胞表達(dá)；與對(duì)照組相比,PP2Ac蛋白表達(dá)量并無(wú)顯著改變,但是PP2Ac硝基化水平明顯升高;和其他翻譯后修飾相比,PP2Ac硝基化可明顯上調(diào)PP2A磷酸酶活性(P0.05)。結(jié)論P(yáng)P2A激活可破壞內(nèi)皮細(xì)胞穩(wěn)定性,促進(jìn)EndMT的發(fā)生,抑制PP2A活性可減弱EndMT效應(yīng)。PP2Ac硝基化是內(nèi)皮細(xì)胞PP2A活性增強(qiáng)的主要原因,提示PP2Ac硝基化在內(nèi)皮細(xì)胞轉(zhuǎn)分化中發(fā)揮重要的促進(jìn)作用。第三部分PP2Ac Y127硝基化在EndMT中的作用研究目的前期研究表明蛋白絲/蘇氨酸磷酸酶2A (PP2A)的催化亞單位C (PP2Ac)硝基化可激活PP2A并能下調(diào)緊密連接蛋白Occludin絲/蘇氨酸位點(diǎn)磷酸化,從而參與內(nèi)皮細(xì)胞緊密連接功能調(diào)控。本部分研究探討PP2Ac酪氨酸硝基化具體位點(diǎn)及核心位點(diǎn),并闡明該位點(diǎn)是腎小管管周毛細(xì)血管EndMT的關(guān)鍵調(diào)控位點(diǎn),參與進(jìn)行性管周毛細(xì)血管萎縮,為探索防治腎間質(zhì)纖維化提供潛在的治療靶點(diǎn)和干預(yù)策略。方法 (1)體外構(gòu)建硝基化反應(yīng)體系,用過(guò)氧化亞硝酸鹽(Peroxynitrite)刺激重組PP2Ac蛋白,western blot收集對(duì)照組和刺激組中的蛋白行質(zhì)譜檢測(cè),分析PP2Ac酪氨酸發(fā)生硝基化的具體位點(diǎn)。(2)對(duì)上述硝基化位點(diǎn)進(jìn)行計(jì)算機(jī)模擬分析以明確各位點(diǎn)空間暴露程度從而確定調(diào)控PP2A活性的核心位點(diǎn)。同時(shí)構(gòu)建以酪氨酸硝基化位點(diǎn)為中心的底物模擬多肽,偶聯(lián)TAT穿膜肽,采用化學(xué)合成法獲得融合多肽。(3)為了驗(yàn)證多肽是否能夠順利穿透進(jìn)入內(nèi)皮細(xì)胞,高效運(yùn)載PP2Ac活性片段,我們運(yùn)用FITC標(biāo)記該多肽。將多肽與HUVECs共培養(yǎng)72小時(shí)后,熒光顯微鏡下觀察該多肽細(xì)胞內(nèi)熒光強(qiáng)度并采用CCK8法篩選最佳細(xì)胞藥物濃度。(4)體外實(shí)驗(yàn)驗(yàn)證上述多肽對(duì)EndMT的干預(yù)效應(yīng),進(jìn)一步明確PP2Ac核心位點(diǎn)。結(jié)果(1)質(zhì)譜結(jié)果顯示,對(duì)照組僅檢測(cè)出一個(gè)硝基化位點(diǎn)Y218,實(shí)驗(yàn)組PP2Ac硝基化位點(diǎn)增加至六個(gè)：Y284/267/265/218/130/127.計(jì)算機(jī)模擬分析顯示,在這六個(gè)位點(diǎn)中,Y127在蛋白質(zhì)空間結(jié)構(gòu)、相鄰氨基酸電荷分布上具有更穩(wěn)定的空間表位優(yōu)勢(shì),在PP2Ac活化過(guò)程中可能起核心作用。(2)以構(gòu)建的TAT-127WT融合多肽為例,攜帶熒光標(biāo)記的多肽可滲透進(jìn)內(nèi)皮細(xì)胞且可持續(xù)到實(shí)驗(yàn)終點(diǎn)時(shí)間72h,對(duì)內(nèi)皮細(xì)胞具良好的穿透效率。CCK8法檢測(cè)不同濃度下該多肽的細(xì)胞毒性,結(jié)果表明在5-10uM濃度下該多肽不僅具有良好的穿透性,對(duì)細(xì)胞的毒性作用最低。因此,在后續(xù)的細(xì)胞實(shí)驗(yàn)中選擇10uM進(jìn)行研究。(3)以各位點(diǎn)為中心構(gòu)建合成的可穿透性底物模擬多肽抑制TGF-β1誘導(dǎo)的EndMT效應(yīng)依次為：Y127Y265Y130284Y267。結(jié)論酪氨酸127是PP2Ac硝基化及PP2A活化的關(guān)鍵位點(diǎn),以該位點(diǎn)為核心構(gòu)建的底物模擬多肽可有效抑制EndMT,可能是管周毛細(xì)血管病變、間質(zhì)纖維化治療的潛在生物靶點(diǎn)。第四部分TAT-Y127WT對(duì)EndMT的體內(nèi)外干預(yù)研究目的前期研究表明,PP2Ac Y127是PP2A全酶活性的核心位點(diǎn),以PP2Ac Y127為中心構(gòu)建的底物模仿多肽較其他位點(diǎn)具有更顯著的抑制EndMT效應(yīng)。本部分研究進(jìn)一步探討TAT-Y127WT及其對(duì)照多肽TAT-Y127Scr對(duì)內(nèi)皮細(xì)胞轉(zhuǎn)分化的體內(nèi)外干預(yù)效應(yīng),為腎間質(zhì)纖維化的治療提供實(shí)驗(yàn)依據(jù)和策略。方法(1)合成以PP2Ac Y127為中心的底物模擬多肽(TAT-127WT)和對(duì)照多肽(TAT-127Scr),預(yù)處理HUVECs 30min,給予TGF-β1 (10ng/ml)刺激72h, western blot檢測(cè)a-SMA、VE-cadherin蛋白表達(dá)變化及PP2A底物Occludin磷酸化水平。(2)運(yùn)用活體動(dòng)物成像技術(shù),在多肽注射前、注射后30min、4h及24h觀察其在體內(nèi)的代謝情況。同時(shí)在相應(yīng)時(shí)間點(diǎn)取出重要器官和組織進(jìn)行示蹤。(3)尾靜脈聯(lián)合腹腔注射(5 nmol/g,術(shù)前一次、術(shù)后1次／天),2周后觀察該多肽對(duì)UUO模型鼠腎臟管周毛細(xì)血管內(nèi)皮細(xì)胞EndMT的干預(yù)效應(yīng),免疫組化觀察α-SMA、Vimentin沉積情況。結(jié)果(1) Western blot結(jié)果顯示TAT-127WT組較TGF-β1組、TAT-127Scr組,a-SMA表達(dá)顯著下調(diào),而VE-cadherin和occludin磷酸化水平顯著上調(diào),差異具有統(tǒng)計(jì)學(xué)意義(P0.05) 。 (2) TAT-127WT在體內(nèi)的代謝速度較快,4h后不足10%,24h后完全從體內(nèi)代謝。肝臟和腎臟藥物的富集程度較高,心、肺、脾臟程度低。檢測(cè)血液中熒光強(qiáng)度進(jìn)一步證明該融合多肽的半衰期大約為2.5h。(3)較對(duì)照組,經(jīng)Y127WT干預(yù)后,腎組織微血管密度上調(diào),具有一定EndMT抑制效應(yīng)。免疫組化結(jié)果顯示,干預(yù)組能夠部分減少α-SMA、Vimentin在腎間質(zhì)中的表達(dá)和積聚,差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。結(jié)論通過(guò)抑制EndMT,以PP2Ac Y127為核心的底物模擬多肽對(duì)UUO小鼠腎小管管周毛細(xì)血管病變具有一定的改善作用,為腎間質(zhì)纖維化治療提供了新的思路。
[Abstract]:Objective intermesenchymal transition (EndMT) is an important source of newly discovered renal interstitial myofibroblasts. The mechanism is closely related to the close connection of endothelial cells to the loss of function. The study shows that the tyrosine loci nitro of C (PP2Ac) C (PP2Ac) is catalyzed by protein filament / threonine phosphatase (PP2A). The activation of PP2A and the downregulation of the phosphorylation of the tightly connected connexin (Occludin) silk / threonine loci are involved in the functional regulation of the tight junction of endothelial cells. This study explores the effect of nitroylation of PP2Ac tyrosine core sites on the tight junction of endothelial cells, and elucidates that this site is an important regulation of the capillary EndMT in the tubules of the renal tubules. The molecule, participating in progressive renal interstitial fibrosis, provides experimental basis for the exploration of new therapeutic targets for the prevention and treatment of renal interstitial fibrosis. Methods the nitro reaction system was constructed in vitro, the recombinant PP2Ac protein was stimulated with nitrite (Peroxynitrite), and Western blotting was used to collect the protein mass spectrometry in the control group and the stimulation group. Analysis of the specific loci of nitration of PP2Ac tyrosine. Computer simulation analysis of the nitrocellulose sites to determine the degree of space exposure to determine the core sites to regulate the activity of PP2A. At the same time, we construct, synthesize the substrate mimic and control peptides centered on the core site, and verify the polypeptide in vitro. The intervention of EndMT in vivo was first used in vivo imaging to detect the distribution and metabolic state of peptides in mice. Then, the intervention effect of the polypeptide on the peripheral capillary EndMT in the UUO mouse model was observed by intraperitoneal polypeptide injection with the tail vein. The loci have Y284/267/265/218/130/127. In these six loci, Y127 has a more stable spatial epitope advantage in the spatial structure of protein, the distribution of adjacent amino acids, and plays a core role in the process of PP2Ac activation. The penetrable substrate mimic peptide and the control polypeptide can inhibit the TGF- beta 1 induced by the 127 loci as the center. In EndMT. in vitro test, peptides have high drug accumulation in the liver and kidney. Compared with the control polypeptide, the substrate mimic peptide can obviously inhibit the dephosphorylation of Occludin, reduce the occurrence of EndMT, stabilize the peripheral capillary structure and partially alleviate the renal interstitial fibrosis. Conclusion the analog peptide of substrate with PP2Ac Y127 as the core can be effectively dried. Preendothelial cell transdifferentiation is a potential biological target for the treatment of renal interstitial fibrosis. The first part of the perivascular capillary EndMT is an important part of renal interstitial fibrosis in order to explore the evidence of EndMT in primary and secondary renal diseases and UUO animal models, and to observe the transformation of growth factor beta 1 (Transforming growth factor be). TA 1, TGF- beta 1) promote the effect of mesenchymal transition in endothelial cells and explore the endotheliogenesis of myofibroblast in renal interstitial fibrosis. Methods the renal biopsy specimens of primary renal disease (IgA nephropathy) and secondary renal diseases (lupus nephritis and diabetic nephropathy) were selected, and CD31 and myofibroblast specific markers of vascular endothelial cells were used. The specific marker a-SMA was detected by continuous slice immunofluorescence, and the co confocal three-dimensional structural reconstruction was used in the positive region to clear the co expression of CD31+a-SMA+. In the body, the C57 mouse unilateral ureteral obstruction nephropathy model was used, and the co expression of CD31+a-SMA+ was observed by the fluorescence double labeling combined with three-dimensional reconstruction. The in vitro experiment was carried out with TGF- beta 1 (lOng/ml). The human umbilical vein endothelial cells (72h) were stimulated to establish a EndMT cell model. The morphological changes of the cells were observed under the microscope. The expression of vascular endothelial cadherin (VE-cadherin, endothelial specific marker) and smooth muscle actin (a-SMA) was detected by immunofluorescence and Western blot. The results were compared with those in IgA nephropathy, lupus nephritis, diabetic nephropathy and UU. O animal model renal interstitial fibrosis area, abnormal expansion or reduction of perivascular capillary tube; confocal microscope observation found that CD31 and alpha -SMA co expression increased, the difference was statistically significant (P0.05); the co expression part of the vascular three-dimensional reconstruction, the expression of partial endothelial cells can express alpha -SMA. in vitro experiment, TGF- beta 1 prickly After stimulated 72h, the morphology of endothelial cells was transformed from paving stone to spindle type, and the distance between cells and cells increased obviously. Compared with the control group, the expression of myofibroblast marker alpha -SMA increased significantly, while the expression of VE-cadherin was down down, the difference was statistically significant (P0.05). Conclusion endothelial cells were in primary and secondary renal diseases. Mesenchymal stromal differentiation is an important component of myofibroblast in renal interstitial fibrosis. Endothelial cells are stimulated by fibrotic factors. Cell phenotypes and functional properties can be transformed into mesenchymal cells. Second the activation of PP2A and the effect of PP2Ac nitro on EndMT, the purpose of this study is to explore the activation of endothelial cells PP2A and PP2Ac nitration. The influence method of EndMT process (1) extracts the total protein of the renal tissue protein in UUO mice and the time point of different TGF- beta 1 at different time points. Then the activity changes of PP2A are monitored with silk / threonine protein phosphatase activity kit. (2) PP2A specificity inhibitor, Okadaic acid, OA, is used to observe the TGF The expression of alpha -SMA, VE-cadherin protein expression and PP2A substrate close connexin Occludin phosphorylation level under the stimulation of beta 1; (3) PP2Ac is the active subunit of PP2A, using immunohistochemistry and immunofluorescence to locate the expression of PP2Ac in the renal tissue, and to detect the changes of PP2Ac post-translational modification of endothelial cells stimulated by TGF- beta 1 by immunofluorescence. The effect of PP2A phosphatase activity. (1) compared with the control group, the activity of PP2A increased gradually and reached the peak at UUO-14d with the prolongation of the obstruction time. In vitro, under the stimulation of TGF- beta 1, the activity of PP2A increased in 15min and increased significantly in 60min (P0.05). (2) Western blot results showed the TGF- beta 1 group, the OA intervention group expressed a significant expression. The expression of VE-cadherin was significantly up-regulated, and immunoprecipitation showed that the phosphorylation level of the close connexin Occludin in normal HUVE cells was maintained at a high level. The phosphorylation level of TGF- beta 1 stimulated 72h significantly decreased, and Occludin phosphorylation decreased partially after OA pretreatment (P0.05). (3) immunofluorescence detection showed that PP2Ac was mainly in the glomerulus Compared with the control group, the expression of PP2Ac protein was not significantly changed, but the level of PP2Ac nitrochemical increased significantly. Compared with other post-translational modifications, PP2Ac nitro could obviously increase the activity of PP2A phosphatase (P0.05). Conclusion PP2A activation could destroy the stability of endothelial cells and promote the occurrence of EndMT. Inhibition of PP2A activity can weaken the EndMT effect of.PP2Ac nitration is the main reason for the enhancement of PP2A activity in endothelial cells, suggesting that PP2Ac nitration plays an important role in the transformation of endothelial cells. Third the role of PP2Ac Y127 nitration in EndMT The nitrification of C (PP2Ac) can activate PP2A and reduce the phosphorylation of the close connexin Occludin / threonine site, thus participating in the regulation of the tight junction function of endothelial cells. This part studies the position and core loci of PP2Ac tyrosine nitroylation, and clarifies that this site is the key regulation of the EndMT in the capillary tube of the renal tubule. Loci, participating in progressive perivascular capillary atrophy, provides potential therapeutic targets and intervention strategies for the prevention and treatment of renal interstitial fibrosis. Methods (1) the nitro reaction system was constructed in vitro, the recombinant PP2Ac protein was stimulated with nitrite (Peroxynitrite), and Western blot was used to collect protein mass spectrometry in the control group and the stimulation group. Analysis of the specific loci of nitro formation of PP2Ac tyrosine. (2) computer simulation analysis of the nitro loci was carried out to determine the degree of space exposure to determine the core loci that regulate the activity of PP2A. At the same time, the substrate simulated polypeptides with tyrosine nitro site as the center, coupled with TAT membrane peptide, were obtained by chemical synthesis. (3) (3) in order to verify whether the polypeptide can penetrate into the endothelial cells successfully and efficiently carry the active fragment of the polypeptide, we use FITC to mark the polypeptide. After co culture of the polypeptide and HUVECs for 72 hours, the fluorescence intensity of the polypeptide cell is observed under the fluorescence microscope and the best cell drug concentration is screened by CCK8 method. (4) in vitro experiment The interference effect of the polypeptide to EndMT was verified and the core site of PP2Ac was further clarified. Results (1) the results of mass spectrometry showed that only a nitrochemical site Y218 was detected in the control group, and the nitrochemical site of PP2Ac in the experimental group was increased to six: Y284/267/265/218/130/127. computer simulation analysis showed that in the six loci, Y127 was in the protein space junction. Structure, the adjacent amino acid charge distribution has a more stable spatial epitope advantage and may play a core role in the process of PP2Ac activation. (2) taking the constructed TAT-127WT fusion peptide as an example, the polypeptide carrying fluorescent labeled peptides can permeate into the endothelial cells and continue to the end of the experiment at 72h, which has good penetration efficiency.CCK8 assay for endothelial cells. The cytotoxicity of the polypeptide was measured at different concentrations. The results showed that the polypeptide not only had good penetration but the lowest toxicity to the cells at the concentration of 5-10uM. Therefore, 10uM was selected in the subsequent cell experiments. (3) a synthetic penetrable substrate analogue polypeptide was constructed to inhibit the EndMT induced by TGF- beta 1. The effect is: Y127Y265Y130284Y267. conclusion tyrosine 127 is the key site of PP2Ac nitration and PP2A activation. The substrate mimic peptide constructed with this site can effectively inhibit EndMT. It may be a potential biological target for the treatment of interstitial fibrosis and interstitial fibrosis. The fourth part of TAT-Y127WT is a pre research on the body and body of EndMT in the body. The previous study showed that PP2Ac Y127 was the core site of the whole enzyme activity of PP2A, and that the substrate constructed by PP2Ac Y127 as the center was more significant than the other loci in inhibiting the EndMT effect. This part of the study further explored the effect of TAT-Y127WT and its control polypeptide TAT-Y127Scr on the transdifferentiation of endothelial cells in vitro and in vivo. The treatment of interstitial fibrosis provides experimental basis and strategy. Methods (1) synthesize PP2Ac Y127 centered substrate analog peptide (TAT-127WT) and control polypeptide (TAT-127Scr), pretreat HUVECs 30min, give TGF- beta 1 (10ng/ml) to stimulate 72h, Western blot detection a-SMA, protein expression and substrate phosphorylation level. (2) In vivo animal imaging technique was used to observe the metabolism in the body before injection of polypeptide, 30min, 4H and 24h after injection. At the same time, the important organs and tissues were taken out to trace. (3) the tail vein was injected with intraperitoneal injection (5 nmol/g, one time before, 1 days after operation), and after 2 weeks, the polypeptide was observed on the kidney tube Zhou Maoxi of UUO model rat Intervention effect of vascular endothelial cell EndMT, immunohistochemical observation of alpha -SMA, Vimentin deposition. Results (1) Western blot results showed that TAT-127WT group was more than TGF- beta 1 group, TAT-127Scr group, a-SMA expression was significantly down, and VE-cadherin and occludin phosphorylation level was significantly up, difference was statistically significant (2) in vivo The metabolic rate was faster, less than 10% after 4h, and after 24h, the concentration of liver and kidney was high, heart, lung and spleen were low. The fluorescence intensity in the blood showed that the half-life of the fusion polypeptide was about 2.5h. (3) compared with the control group. After the Y127WT dry prognosis, the density of the renal tissue was up regulated, with a certain EndMT suppression. The results of immuno histochemistry showed that the intervention group could partially reduce the expression and accumulation of alpha -SMA, Vimentin in the renal interstitium, and the difference was statistically significant (P0.05). Conclusion by inhibiting EndMT, the analog peptide of the substrate with PP2Ac Y127 as the core has a certain improvement in the renal tubule Zhou Mao fine vascular lesions of UUO mice, and it is the renal interstitium. The treatment of fibrosis provides a new way of thinking.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：R692

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1 鄧元俊;PP2Ac Y127位點(diǎn)硝基化在腎小管管周毛細(xì)血管內(nèi)皮細(xì)胞轉(zhuǎn)分化中的作用及干預(yù)研究[D];華中科技大學(xué);2016年

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