沉默COX-2对非酒精性脂肪肝细胞凋亡及神经酰胺水平的影响

发布时间：2018-01-05 11:32

本文关键词：沉默COX-2对非酒精性脂肪肝细胞凋亡及神经酰胺水平的影响　出处：《南华大学》2016年硕士论文　论文类型：学位论文

【摘要】：研究背景与目的:神经酰胺(Ceramide,Cer)作为细胞内第二信使分子,广泛参与细胞增殖、分化、生长抑制和凋亡等多种细胞活动,是重要的促凋亡因子,参与非酒精性脂肪病(Non-alcoholic fatty liver disease,NAFLD)的发生发展。环氧化酶-2(Cyclooxygenase-2,COX-2)是前列腺素合成限速酶,由各种刺激因子诱导产生,参与炎症、纤维化和肿瘤等病理过程,可促进肝细胞凋亡,加重肝纤维化。因此,本研究从神经酰胺这一新视角探讨COX-2促进肝细胞凋亡参与NAFLD形成的机制,为研究NAFLD机制及防治提供新思路和寻找新的药物治疗靶点。方法:48只雄性SD大鼠适应性培养1周后,完全随机分为4组:正常对照组、NAFLD模型组、空质粒组、COX-2shRNA组,每组12只。除对照组外,饲喂高脂饲料进行造模,空质粒组和COX-2shRNA组大鼠于造模第一周开始,分别尾静脉注射空质粒载体和腺病毒(PBS稀释),剂量为1×109 pfu/只,每周1次,共12周。苏木精-伊红染色法(Hematoxylin-eosin staining,HE stain)检测大鼠肝脏脂肪变性、纤维化程度和炎症活动度;油红O染色检测肝细胞脂肪变性程度;Masson染色检测大鼠肝脏胶原面积;实时荧光定量PCR(Real Time-PCR,RT-PCR)检测肝组织中COX-2及α-SMAmRNA表达水平;TUNEL染色检测大鼠肝细胞凋亡程度;高效液相色谱法(High performance liquid chromatography,HPLC)检测肝脏神经酰胺水平。结果:1.COX-2shRNA改善NAFLD大鼠肝脏脂肪变性、胶原沉积、炎性浸润及纤维化程度。2.COX-2shRNA组大鼠肝脏组织中COX-2mRNA表达水平低于NAFLD模型组及空质粒组(P0.001)。3.COX-2shRNA组大鼠肝脏组织中α-SMAmRNA表达水平低于NAFLD模型组及空质粒组(P0.001)。4.正常组、NAFLD模型、空质粒组及COX-2 shRNA组大鼠肝组织细胞凋亡指数分别为(1.80±0.94)%、(6.47±2.53)%、(4.93±1.87)%、(3.93±1.67)%。COX-2shRNA组凋亡指数低于NAFLD模型组及空质粒组(P0.001)。5.COX-2 shRNA组大鼠肝组织神经酰胺水平低于NAFLD模型组及空质粒组(P0.001)。结论:1.沉默COX-2抑制NAFLD肝细胞凋亡。2.沉默COX-2降低NAFLD肝组织中神经酰胺水平。
[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 伪 -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 伪 -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 卤0.94%, 6.47 卤2.53% respectively. 4.93 卤1.87%. The apoptotic index in the group of 3.93 卤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.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R575.5

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