本文關(guān)鍵詞：沉默COX-2對(duì)非酒精性脂肪肝細(xì)胞凋亡及神經(jīng)酰胺水平的影響　出處：《南華大學(xué)》2016年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 環(huán)氧合酶2 神經(jīng)酰胺 非酒精性脂肪性肝病 細(xì)胞凋亡

【摘要】：研究背景與目的:神經(jīng)酰胺(Ceramide,Cer)作為細(xì)胞內(nèi)第二信使分子,廣泛參與細(xì)胞增殖、分化、生長(zhǎng)抑制和凋亡等多種細(xì)胞活動(dòng),是重要的促凋亡因子,參與非酒精性脂肪病(Non-alcoholic fatty liver disease,NAFLD)的發(fā)生發(fā)展。環(huán)氧化酶-2(Cyclooxygenase-2,COX-2)是前列腺素合成限速酶,由各種刺激因子誘導(dǎo)產(chǎn)生,參與炎癥、纖維化和腫瘤等病理過程,可促進(jìn)肝細(xì)胞凋亡,加重肝纖維化。因此,本研究從神經(jīng)酰胺這一新視角探討COX-2促進(jìn)肝細(xì)胞凋亡參與NAFLD形成的機(jī)制,為研究NAFLD機(jī)制及防治提供新思路和尋找新的藥物治療靶點(diǎn)。方法:48只雄性SD大鼠適應(yīng)性培養(yǎng)1周后,完全隨機(jī)分為4組:正常對(duì)照組、NAFLD模型組、空質(zhì)粒組、COX-2shRNA組,每組12只。除對(duì)照組外,飼喂高脂飼料進(jìn)行造模,空質(zhì)粒組和COX-2shRNA組大鼠于造模第一周開始,分別尾靜脈注射空質(zhì)粒載體和腺病毒(PBS稀釋),劑量為1×109 pfu/只,每周1次,共12周。蘇木精-伊紅染色法(Hematoxylin-eosin staining,HE stain)檢測(cè)大鼠肝臟脂肪變性、纖維化程度和炎癥活動(dòng)度;油紅O染色檢測(cè)肝細(xì)胞脂肪變性程度;Masson染色檢測(cè)大鼠肝臟膠原面積;實(shí)時(shí)熒光定量PCR(Real Time-PCR,RT-PCR)檢測(cè)肝組織中COX-2及α-SMAmRNA表達(dá)水平;TUNEL染色檢測(cè)大鼠肝細(xì)胞凋亡程度;高效液相色譜法(High performance liquid chromatography,HPLC)檢測(cè)肝臟神經(jīng)酰胺水平。結(jié)果:1.COX-2shRNA改善NAFLD大鼠肝臟脂肪變性、膠原沉積、炎性浸潤(rùn)及纖維化程度。2.COX-2shRNA組大鼠肝臟組織中COX-2mRNA表達(dá)水平低于NAFLD模型組及空質(zhì)粒組(P0.001)。3.COX-2shRNA組大鼠肝臟組織中α-SMAmRNA表達(dá)水平低于NAFLD模型組及空質(zhì)粒組(P0.001)。4.正常組、NAFLD模型、空質(zhì)粒組及COX-2 shRNA組大鼠肝組織細(xì)胞凋亡指數(shù)分別為(1.80±0.94)%、(6.47±2.53)%、(4.93±1.87)%、(3.93±1.67)%。COX-2shRNA組凋亡指數(shù)低于NAFLD模型組及空質(zhì)粒組(P0.001)。5.COX-2 shRNA組大鼠肝組織神經(jīng)酰胺水平低于NAFLD模型組及空質(zhì)粒組(P0.001)。結(jié)論:1.沉默COX-2抑制NAFLD肝細(xì)胞凋亡。2.沉默COX-2降低NAFLD肝組織中神經(jīng)酰胺水平。
[Abstract]:Background & AIM: Ceramide-Ceramide-Ceramide-Ceramide-Ceramide (Ceramide-Ceramide-Ceramide-Ceramide-Ceramide-Ceramide-Ceramide-Ceramide-Ceramide-Ceramide-Ceram@@. It is an important apoptosis-promoting factor and participates in non-alcoholic fatty liver disease. Cyclooxygenase-2Cyclooxygenase-2COX-2) is a rate-limiting enzyme for prostaglandin synthesis induced by various stimulators and involved in inflammation. Pathological processes such as fibrosis and tumor can promote hepatocyte apoptosis and aggravate hepatic fibrosis. In this study, we explored the mechanism of COX-2 promoting hepatocyte apoptosis in the formation of NAFLD from the perspective of ceramide. Methods 48 male Sprague-Dawley rats were cultured for one week and were randomly divided into 4 groups: normal control group. NAFLD model group, empty plasmid group, COX-2shRNA group, 12 rats in each group. At the beginning of the first week of modeling, empty plasmid vector and adenovirus were injected into the tail vein of rats in the empty plasmid group and the COX-2shRNA group, respectively. The dose was 1 脳 109pfuper rat. Hematoxylin-eosin stainingstainin (Hematoxylin-eosin staining) was used to detect hepatic steatosis in rats once a week for 12 weeks. Degree of fibrosis and inflammation; The degree of steatosis in hepatocytes was detected by oil red O staining. The area of liver collagen was detected by Masson staining. The expression of COX-2 and 偽 -SMA mRNA in liver tissue was detected by real-time quantitative PCR(Real Time-PCR- RT-PCR. The degree of hepatocyte apoptosis was detected by TUNEL staining. High performance liquid chromatography. Results the liver ceramide level was determined by HPLC.The results 1. 1. COX-2shRNA improved hepatic steatosis and collagen deposition in NAFLD rats. Degree of inflammatory infiltration and Fibrosis. 2. The expression of COX-2mRNA in liver of rats in COX-2 shRNA group was lower than that in NAFLD model group and empty plasmid group (P0.001). 3. The expression of 偽 -SMA mRNA in liver tissue of rats in COX-2shRNA group was lower than that in NAFLD model group and empty plasmid group (P0.001n.4. normal group). The apoptosis index of hepatocytes in NAFLD model, empty plasmid group and COX-2 shRNA group was 1.80 鹵0.94%, 6.47 鹵2.53% respectively. 4.93 鹵1.87%. The apoptotic index in the group of 3.93 鹵1.67k.COX-2shRNA was lower than that in the NAFLD model group and the blank plasmid group (P0.001). 5.The level of ceramide in liver tissue in COX-2 shRNA group was lower than that in NAFLD model group and empty plasmid group (P0.001). Conclusion: 1. Silencing COX-2 inhibits apoptosis of NAFLD hepatocytes. 2. Silencing COX-2 reduces ceramide level in NAFLD liver tissue.
【學(xué)位授予單位】：南華大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R575.5

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