本文選題：可溶性環(huán)氧化物水解酶抑制劑 + 內(nèi)皮-間質(zhì)轉(zhuǎn)化��； 參考：《鄭州大學(xué)》2017年碩士論文

【摘要】：背景心肌纖維化(cardinal fibrosis,MF)是指在多種病理因素作用下心肌成纖維細(xì)胞的過度增殖、膠原纖維的過度沉積、比例失調(diào)及異常分布所致的心臟間質(zhì)重構(gòu)。MF是心臟對(duì)各種刺激產(chǎn)生的適應(yīng)性反應(yīng),也是心肌缺血、心肌病、心力衰竭、糖尿病、高血壓、肺動(dòng)脈高壓等多種心血管疾病發(fā)展至一定階段共同的病理生理改變。多種機(jī)制參與心肌纖維化的進(jìn)程,包括腎素-血管緊張素-醛固酮系統(tǒng)(renin-angiotensin-aldosterone system,RAAS)、內(nèi)皮素(endothelin,ET)、核因子κB(nuclear factorκB,NF-κB)、一氧化氮(nitric oxide,NO)、轉(zhuǎn)化生長(zhǎng)因子-β(transforming growth factor-β1,TGF-β)等。減少心肌重構(gòu)能夠改善患者的預(yù)后,因此尋找特異性的抗心肌纖維化藥物,減少心肌重構(gòu)成為了當(dāng)前研究的熱點(diǎn)。近些年來,隨著對(duì)心肌纖維化發(fā)生機(jī)制認(rèn)識(shí)的逐步深入,內(nèi)皮-間質(zhì)轉(zhuǎn)化(endothelial mesenchymal transition,EndMT)在心肌纖維化中的作用受到越來越多研究者的重視。內(nèi)皮-間質(zhì)轉(zhuǎn)化屬于上皮-間質(zhì)轉(zhuǎn)化(Epithelial mesenchymal transition,EMT)的一種特殊類型,是內(nèi)皮細(xì)胞在多種因素刺激下逐漸失去其形態(tài)和功能,向間充質(zhì)細(xì)胞表型轉(zhuǎn)化并獲得增殖、遷移及合成膠原等功能的過程。TGF-β信號(hào)、Notch信號(hào)、Wnt信號(hào)、MicroRNAs及一些炎癥因子等可能參與EndMT的調(diào)節(jié),其中TGF-β1/Smads信號(hào)通路是研究最為深入的一種。環(huán)氧化物水解酶(epoxide hydratase,EH)普遍存在于哺乳動(dòng)物體內(nèi),作用于心血管疾病的多個(gè)環(huán)節(jié),具有舒張血管、抗炎、刺激血管形成等作用�？扇苄原h(huán)氧化物水解酶抑制劑(soluble epoxied hydrolase inhibitor,sEHI)是EH的抑制劑,參與抑制多環(huán)芳烴環(huán)氧化物、環(huán)氧二十碳三烯酸(epoxyeicosatrienoic acid,EET)等環(huán)氧化物的代謝,它具有抗心肌纖維化、調(diào)節(jié)血脂、抗高血壓及減少心肌缺血再灌注損傷等作用。然而sEHI是否可以通過抑制EndMT發(fā)揮抗心肌纖維化作用及其抗纖維化作用是否是通過抑制TGF-β1/Smads信號(hào)通路來實(shí)現(xiàn)尚無人報(bào)道。目的本研究采用TGF-β1誘導(dǎo)人臍靜脈內(nèi)皮細(xì)胞(human umbilical vein epithelial cell,HUVEC-12)發(fā)生內(nèi)皮-間質(zhì)轉(zhuǎn)化,并給予不同劑量的t-AUCB進(jìn)行干預(yù)。通過CCK-8法觀察細(xì)胞活性,real time RT-PCR檢測(cè)內(nèi)皮、間質(zhì)標(biāo)志物和TGF-β1/Smads下游轉(zhuǎn)錄因子的基因表達(dá),光鏡觀察HUVEC-12細(xì)胞形態(tài),Western blot檢測(cè)Smad2/3的磷酸化水平。初步探討t-AUCB抗纖維化作用與TGF-β1/Smads信號(hào)通路的相關(guān)性,為t-AUCB抗心肌纖維化的應(yīng)用提供細(xì)胞實(shí)驗(yàn)依據(jù)。材料與方法人臍靜脈內(nèi)皮細(xì)胞株HUVEC-12購(gòu)自YRGene(NC006),在37℃、5%CO2的培養(yǎng)箱中培養(yǎng)。實(shí)驗(yàn)前將細(xì)胞置于無血清培養(yǎng)基中進(jìn)行6h的饑餓處理。然后將其分為四組:對(duì)照組(無血清DMEM培養(yǎng)液持續(xù)培養(yǎng))、TGF-β1組(含10μg/L TGF-β1的無血清DMEM培養(yǎng)液培養(yǎng)72h)、t-AUCB干預(yù)組(先采用含50μmol/L t-AUCB無血清DMEM培養(yǎng)液預(yù)保護(hù)40min,后采用含10μg/L TGF-β1及50μmol/L t-AUCB的無血清DMEM培養(yǎng)液共同培養(yǎng)72h)、t-AUCB(含50μmol/L t-AUCB無血清DMEM培養(yǎng)液持續(xù)培養(yǎng)72h)。各組細(xì)胞培養(yǎng)24h時(shí),采用Western blot檢測(cè)各組細(xì)胞Smad2/3的磷酸化水平。各組細(xì)胞培養(yǎng)72h時(shí),應(yīng)用細(xì)胞毒性試劑盒(cell counting kit-8,CCK-8)檢測(cè)細(xì)胞活性;光鏡觀察細(xì)胞形態(tài)的變化;實(shí)時(shí)熒光定量PCR(Real-time PCR,RT-PCR)檢測(cè)內(nèi)皮標(biāo)志物CD31及間質(zhì)標(biāo)志物collagen I、collagenIII、vimentin的基因表達(dá)和TGF-β1/Smads信號(hào)通路中下游轉(zhuǎn)錄因子snail 1、twist 1、twist 2、ZEB1的基因表達(dá)。結(jié)果(1)TGF-β1(10μg/L)和/或t-AUCB(1、10、50μmol/L)對(duì)細(xì)胞活性影響差異無統(tǒng)計(jì)學(xué)意義(P均0.05)。(2)real time RT-PCR結(jié)果提示,TGF-β1(10μg/L)誘導(dǎo)培養(yǎng)HUVEC-12細(xì)胞72 h后,內(nèi)皮細(xì)胞標(biāo)記物CD31的基因表達(dá)顯著下調(diào),間質(zhì)標(biāo)記物collagen I、collagen III、vimentin的基因表達(dá)則顯著上調(diào);TGF-β1(10μg/L)與t-AUCB(1、10、50μmol/L)共同干預(yù)細(xì)胞72 h后可顯著逆轉(zhuǎn)這種現(xiàn)象,且具有濃度依賴性(P均0.05)。(3)光鏡結(jié)果示,對(duì)照組細(xì)胞光鏡下為鵝卵石形,細(xì)胞間連接緊密;TGF-β1干預(yù)組細(xì)胞轉(zhuǎn)變?yōu)楠M長(zhǎng)形,細(xì)胞間隙疏松;TGF-β1與t-AUCB(50μmol/L)聯(lián)合干預(yù)細(xì)胞及單用t-AUCB干預(yù)細(xì)胞72h后,細(xì)胞形態(tài)與對(duì)照組相似。(4)Western blot結(jié)果提示,TGF-β1(10μg/L)干預(yù)HUVEC-12細(xì)胞72h后可顯著提高Smad2/3的磷酸化水平,t-AUCB(50μmol/L)則可顯著逆轉(zhuǎn)上述變化(P均0.05)。(5)real time RT-PCR結(jié)果提示,與對(duì)照組相比,TGF-β1(10μg/L)誘導(dǎo)組細(xì)胞下游轉(zhuǎn)錄因子snail1、twist1、twist2、ZEB1的基因表達(dá)顯著增高;t-AUCB(50μmol/L)與TGF-β1(10μg/L)聯(lián)合干預(yù)時(shí)則可顯著抑制上述基因的表達(dá)(P均0.05)。結(jié)論(1)TGF-β1可誘導(dǎo)的HUVEC-12細(xì)胞發(fā)生間充質(zhì)細(xì)胞表型的轉(zhuǎn)化。(2)t-AUCB對(duì)HUVEC-12細(xì)胞發(fā)生EndMT無明顯影響。(3)t-AUCB可抑制TGF-β1誘導(dǎo)的HUVEC-12細(xì)胞發(fā)生EndMT轉(zhuǎn)化。
[Abstract]:Background myocardial fibrosis (cardinal fibrosis, MF) refers to the excessive proliferation of myocardial fibroblasts under various pathological factors, excessive deposition of collagen fibers, disproportions and abnormal distribution of cardiac interstitial remodeling.MF is the adaptive response of the heart to various stimuli. It is also the myocardial ischemia, cardiomyopathy, heart failure, and diabetes. A variety of cardiovascular diseases, such as disease, hypertension, and pulmonary artery hypertension, develop to a certain stage of common pathophysiology. Many mechanisms are involved in the process of myocardial fibrosis, including the renin angiotensin aldosterone system (renin-angiotensin-aldosterone system, RAAS), endothelin (ET), nuclear factor kappa B (nuclear factor kappa B, NF- kappa B), Nitric oxide (nitric oxide, NO), transforming growth factor - beta (transforming growth factor- beta 1, TGF- beta) and so on. Reducing myocardial remodeling can improve the prognosis of patients. Therefore, finding specific anti myocardial fibrosis drugs and reducing myocardial weight constitute the hot spot of current research. Step by step, the role of endothelial mesenchymal transition (EndMT) in myocardial fibrosis is being paid more attention by more and more researchers. Endothelium transformation belongs to a special type of Epithelial mesenchymal transition (EMT), which is the gradual loss of endothelial cells under a variety of factors. Morphology and function,.TGF- beta signal, Notch signal, Wnt signal, MicroRNAs and some inflammatory factors may be involved in the regulation of EndMT, and TGF- beta 1/Smads signaling pathway is the most in-depth study. Epoxide hydrolase (epoxide hydratase, epoxide hydratase,) EH (soluble epoxied hydrolase inhibitor, sEHI) is an inhibitor of EH, which is an inhibitor of EH, and is involved in the inhibition of polycyclic aromatic epoxides, epoxy twenty carbon three enoic acid (epoxyei). Cosatrienoic acid, EET) and other epoxides metabolism, it has the effect of anti myocardial fibrosis, regulating blood lipid, anti hypertension and reducing myocardial ischemia reperfusion injury. However, whether sEHI can inhibit the effect of anti fibrosis by inhibiting EndMT and its anti fibrosis effect by inhibiting the TGF- beta 1/Smads signaling pathway to achieve this. The purpose of this study was to use TGF- beta 1 to induce endothelial cell transformation in human umbilical vein endothelial cells (human umbilical vein epithelial cell, HUVEC-12), and to give different doses of t-AUCB to intervene. The activity of cells was observed by CCK-8 method. Real time RT-PCR was used to detect the inner skin, interstitial markers and downstream transcription factors. The morphology of HUVEC-12 cells was observed by light microscopy, and the phosphorylation level of Smad2/3 was detected by Western blot. The correlation between the anti fibrosis effect of t-AUCB and the TGF- beta 1/Smads signaling pathway was preliminarily discussed, and the experimental basis for the application of t-AUCB against myocardial fibrosis was provided. Materials and methods of human umbilical vein endothelial cells were purchased from YRGene (NC006). The cells were cultured in the 5%CO2 culture box at 37 degrees C. Before the experiment, the cells were placed in the serum-free medium for 6h starvation. Then they were divided into four groups: the control group (serum-free DMEM culture medium continuous culture), the TGF- beta 1 group (including the serum free DMEM culture solution of 10 mu g/L TGF- beta 1), and the t-AUCB intervention group (50 micron mol/L t-AUCB without serum-free DME). M culture solution preprotected 40min, then cultured 72h containing 10 mu g/L TGF- beta 1 and 50 mu mol/L t-AUCB, and t-AUCB (containing 50 mu t-AUCB serum DMEM culture liquid continuously). The cell activity was detected by cell counting kit-8 (CCK-8); the changes in cell morphology were observed by light microscopy; real-time quantitative PCR (Real-time PCR, RT-PCR) was used to detect the endothelial marker CD31 and the interstitial marker collagen I, collagenIII, the downstream transcription factor 1, and 1 T 2, ZEB1 gene expression. Results (1) there was no significant difference in the effect of TGF- beta 1 (10 mu g/L) and / or t-AUCB (1,10,50 mu mol/L) on cell activity (P 0.05). (2) real time RT-PCR results suggested that TGF- beta 1 (10 micron) induced the cultured cell 72 Agen III, vimentin gene expression was significantly up-regulated, and TGF- beta 1 (10 mu g/L) and t-AUCB (1,10,50 mu mol/L) could significantly reverse this phenomenon after the co intervention of 72 h (P 0.05). (3) the light mirror results showed that the cells in the control group were goose egg shaped and the cells were closely connected; TGF- beta 1 intervention group changed into a narrow shape, Cell space was loose; after TGF- beta 1 and t-AUCB (50 mol/L) intervened cells and single t-AUCB intervention cells 72h, the cell morphology was similar to that of the control group. (4) Western blot results suggested that TGF- beta 1 (10 u g/L) could significantly improve the phosphorylation of Smad2/3. (50 micron) could significantly reverse the above changes (0.05). 5) real time RT-PCR results showed that the gene expression of downstream transcription factor Snail1, Twist1, Twist2 and ZEB1 increased significantly in TGF- beta 1 (10 mu g/L), while t-AUCB (50 mu mol/L) and TGF- beta 1 (10 mu) could significantly inhibit the expression of the above gene (0.05). Conclusion (1) beta 1 induced cell hair. Transformation of the phenotype of mesenchyme cells. (2) t-AUCB has no significant effect on the occurrence of EndMT in HUVEC-12 cells. (3) t-AUCB can inhibit the EndMT transformation of HUVEC-12 cells induced by TGF- beta 1.

【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R542.2

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