【摘要】：目的:1.通過建立非酒精性脂肪肝(NAFLD)體內(nèi)外模型,探討內(nèi)質(zhì)網(wǎng)應(yīng)激(ERS)對NAFLD的影響及機(jī)制。2.以雷公藤紅素干預(yù)NAFLD細(xì)胞模型,探討雷公藤紅素對NAFLD的防治作用,以及對其ERS的影響及可能機(jī)制。方法:1.NAFLD體內(nèi)外模型的建立與鑒定:20只SD雄性大鼠以基礎(chǔ)飼料適應(yīng)性飼養(yǎng)1周后,隨機(jī)分為對照組(Control)和非酒精性脂肪肝組(NAFLD),分別予以基礎(chǔ)飼料及高脂飼料喂養(yǎng)。LO2細(xì)胞分為control組和NAFLD組,分別用基礎(chǔ)培養(yǎng)基和含0.2mmol/L軟脂酸培養(yǎng)基培養(yǎng)。HE染色觀察肝組織的形態(tài)學(xué)改變;油紅O染色分別觀察肝組織和LO2細(xì)胞脂肪蓄積情況;膽固醇(TC)和甘油三酯(TG)試劑盒分別檢測肝組織和LO2細(xì)胞內(nèi)TC、TG含量。2.免疫組織化學(xué)觀測肝組織GRP78蛋白的表達(dá)水平;PCR和Western-blot分別檢測NAFLD細(xì)胞模型中ERS相關(guān)信號(hào)分子ATF6、GRP78、IRE1、SCAP、SREBP-1c和SREBP-2的mRNA及蛋白表達(dá)水平。3.雷公藤紅素干預(yù)實(shí)驗(yàn):將L02細(xì)胞分為control組、NAFLD組、雷公藤紅素低劑量組(Cel 0.5)、雷公藤紅素高劑量組(Cel 1)和辛伐他汀組(SIMVA)。油紅O染色觀察雷公藤紅素干預(yù)后LO2細(xì)胞內(nèi)脂質(zhì)沉積情況;TC和TG試劑盒分別檢測LO2細(xì)胞內(nèi)TC、TG含量;PCR和Western-blot分別檢測雷公藤紅素作用下LO2細(xì)胞中ERS相關(guān)信號(hào)分子ATF6、GRP78、IRE1、SCAP、SREBP-1c和SREBP-2的mRNA以及蛋白表達(dá)水平。結(jié)果:1.NAFLD體內(nèi)外模型的建立與鑒定:he染色顯示control組大鼠肝小葉結(jié)構(gòu)正常;而nafld組大鼠肝細(xì)胞排列紊亂,出現(xiàn)大量脂滴空泡。肝組織和lo2細(xì)胞油紅o染色均顯示control組紅染顆粒少,肝細(xì)胞結(jié)構(gòu)正常,且排列整齊;而nafld組肝細(xì)胞內(nèi)含有大量紅染顆粒且發(fā)生脂肪變性。control組肝組織和lo2細(xì)胞內(nèi)的tc和tg含量均低于nafld組(p0.05)。2.免疫組化結(jié)果顯示nafld組大鼠肝組織grp78蛋白表達(dá)水平高于control組(p0.05);pcr和western-blot結(jié)果顯示nafld組lo2細(xì)胞內(nèi)upr相關(guān)信號(hào)分子atf6,grp78和ire1的mrna和蛋白表達(dá)水平均高于control組(p0.05);nafld組lo2細(xì)胞內(nèi)固醇調(diào)節(jié)級聯(lián)反應(yīng)相關(guān)信號(hào)分子scap,srebp-1c和srebp-2的mrna和蛋白表達(dá)水平均高于control組(p0.05)。3.雷公藤紅素干預(yù)實(shí)驗(yàn):油紅o染色顯示nafld組lo2細(xì)胞內(nèi)含有大量紅染顆粒,而cel0.5μg/ml組、cel1μg/ml組及simva組紅染顆粒較nafld組有不同程度減少。nafld組lo2細(xì)胞tc及tg含量均高于control組(p0.05),cel0.5μg/ml組、cel1μg/ml組及simva組lo2細(xì)胞tc及tg含量均較nafld組有不同程度減少(p0.05)。pcr和western-blot結(jié)果顯示:(1)nafld組lo2細(xì)胞內(nèi)upr相關(guān)信號(hào)分子atf6,grp78和ire1的mrna和蛋白表達(dá)水平均高于control組(p0.05);cel1μg/ml組和simva組lo2細(xì)胞內(nèi)atf6,grp78和ire1的mrna和蛋白表達(dá)水平均低于nafld組(p0.05)。(2)nafld組lo2細(xì)胞內(nèi)固醇調(diào)節(jié)級聯(lián)反應(yīng)相關(guān)信號(hào)分子scap,srebp-1c和srebp-2的mrna和蛋白表達(dá)水平均高于control組(p0.05);cel0.5μg/ml、cel1μg/ml組和simva組lo2細(xì)胞內(nèi)scap,srebp-1c和SREBP-2的mRNA和蛋白表達(dá)水平均低于NAFLD組(P0.05);Cel 1μg/ml組和SIMVA組LO2細(xì)胞內(nèi)SREBP-1c和SREBP-2的mRNA和蛋白表達(dá)水平均低于Cel 0.5μg/ml組(P0.05)。結(jié)論:1.ERS密切參與了NAFLD的脂變過程,在其發(fā)生發(fā)展起著重要作用。2.ERS中的UPR和固醇調(diào)節(jié)級聯(lián)反應(yīng),及相關(guān)的IRE1/XBP-1通路和ATF6通路參與了NAFLD的發(fā)生發(fā)展。3.雷公藤紅素能減輕肝細(xì)胞脂代謝紊亂,改善NAFLD,其機(jī)制可能與緩解肝細(xì)胞的ERS有關(guān)。
[Abstract]:Purpose: 1. Objective To study the effect and mechanism of ER on NAFLD by establishing an external model of non-alcoholic fatty liver (NAFLD). In this paper, the effects of tripterine on NAFLD and its mechanism were discussed. Methods: 1. The establishment and identification of external models in NAFLD were randomly divided into control group and non-alcoholic fatty liver group (NAFLD) after 1 week feeding on basic feed. LO2 cells were divided into control group and NAFLD group, and cultured with basal medium and 0. 2mmol/ L palmitic acid medium respectively. The morphological changes of liver tissue were observed by HE staining; the fat accumulation of liver tissue and LO2 cells were observed by oil-red O staining; TC and TG content in liver tissues and LO2 cells were detected by cholesterol (TC) and triglyceride (TG) kits respectively. The mRNA and protein expression levels of ERS-related signal molecules ATF6, GRP78, IRE1, SCAP, SREBP-1c and SREBP-2 in NAFLD cell model were detected by immunohistochemistry and Western-blot. The results showed that L02 cells were divided into control group, NAFLD group, triptolide low dose group (Cel 0.5), tripterine high dose group (Cel 1) and simvastatin group (SIMVA). The contents of TC and TG in LO2 cells were detected by TC and TG kit respectively, and the ERS-related signal molecules ATF6, GRP78, IRE1 and SCAP in LO2 cells were detected by PCR and Western-blot respectively. mRNA and protein expression levels of SREBP-1c and SREBP-2. Results: 1. The establishment and identification of the outer model in Nafld rats showed that the hepatic lobules were normal in control group. The liver tissue and lo2 cell oil red-o staining showed that the control group had fewer red-stained particles, the structure of hepatocytes was normal, and the arrangement was orderly; and the nafld group hepatocytes contained a large number of red-stained particles and fatty degeneration occurred. The content of tc and DDT in liver tissue and lo2 cells in control group was lower than that of the nafld group (P0.05). The expression level of p78 protein in liver tissue of nafld group was higher than that in control group (P0.05). The mrna and protein expression levels of scap, srebp-1c, and srebp-2 were all higher than that of control group (p0.05). The results showed that there was a large number of red-stained particles in the lo2 cells of the nafld group, while the red-dyed particles of the group were decreased in the group of 0. 5 ug/ ml, 1.mu g/ ml and simva group than in the nafld group. The results showed that: (1) the upr-related signaling molecules in the nafld group of lo2 cells were higher than that of the control group (P0.05), the content of the lo2 cells in the nafld group was higher than that of the control group (P0.05), the content of the lo2 cells in the group of 1.mu. g/ ml and the siva group were reduced to a different extent (P0.05). The expression levels of mrna and protein in p78 and re1 were higher than those in control group (p0.05), and the expression levels of mrna and protein were lower in 1ug/ ml group and simva group, respectively, and the expression level of mrna and protein was lower than that of the nafld group (p0.05). (2) The mRNA and protein expression levels of scap, srebp-1c and srebp-2 in the nafld group lo2 cells were higher than those of control group (P0.05); The mRNA and protein expression levels of SREBP-1c and SREBP-2 in the Cel 1 ug/ ml group and the SIMVA group were lower than that of Cel 0.5ug/ ml group (P0.05). Conclusion: 1. ERS is closely involved in the fat-change process of NAFLD and plays an important role in the development of NAFLD. Triptolide can alleviate hepatocyte lipid metabolism disorder and improve NAFLD, and its mechanism may be related to the response of liver cells to ERS.
【學(xué)位授予單位】：西南醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R575

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