载脂蛋白AI模拟肽D4F对小鼠肝脏脂肪样变和炎性的抑制作用

发布时间：2018-06-10 07:01

本文选题：非酒精性脂肪性肝病 + 肿瘤坏死因子-α　；参考：《泰山医学院》2014年硕士论文

【摘要】：目的本实验通过高脂饲料喂养建立与人类非酒精性脂肪性肝病（NALFD）发病过程相似的载脂蛋白E基因敲除（Apo E-/-）小鼠模型，观察载脂蛋白AI（ApoAI）模拟肽D4F对小鼠肝脏脂质代谢的影响，探讨Apo A模拟肽D4F对小鼠肝脏脂肪样变及炎性的干预作用并进一步探讨其可能的作用机制，，为临床上对NALFD的药物治疗提供依据。方法 24只8周龄雄性Apo E-/-小鼠，经过两周高脂（15.8%）与高胆固醇（1.25%）喂食后随机分为3组(n=8），分别腹腔注射生理盐水(模型组）、紊乱模拟肽sD4F (1mg/kg·d，sD4F组）和Apo AI模拟肽D4F（1mg/kg·d，D4F组）。同周龄雄性C57BL/6J小鼠喂食普通饮食并给予腹腔注射生理盐水作为正常对照组。6周后处死，ELISA法检测Apo E-/-及普通小鼠血浆中炎症因子TNF-α、IL-6的含量，并取小鼠肝脏组织，采用HE染色观察其病理形态的改变，同样采用油红O染色方法评估肝组织脂肪变性程度，免疫组织化学方法检测Apo E-/-及普通小鼠肝组织核因子-κB、TNF-α在肝脏中的表达与分布，同时采用Western Blotting方法检测肝组织中NF-κB、TNF-α的含量。实验数据采用SPSS13.0统计软件包进行统计，两组间比较采用单因素方差检验，实验数据以均数±标准差表示，P0.05认为差异有统计学差异。结果 1.小鼠肝脏组织病理切片结果显示：光学显微镜下可见模型组及sD4F组小鼠的肝脏细胞中出现大量的脂质滴的累积，并伴有不同程度脂肪变性及小叶间炎性细胞的浸润；D4F组小鼠的肝脏细胞脂肪变性程度显著减轻，小叶间炎性细胞浸润明显减少，与对照组小鼠相比无明显差别。 2.对各组小鼠的血清细胞因子的检测结果显示：与对照组小鼠比较，模型组ApoE-/-小鼠血清TNF-α、IL-6的水平增高显著，并有统计学意义（P0.05）；与模型组小鼠相比，D4F组血清TNF-α、IL-6的含量明显下降，分别为模型组的48.6%和63.4%，差异有统计学意义（P0.05）；sD4F组与模型组比较各项指标无统计学差异。 3.免疫组化结果显示：TNF-α在正常组小鼠肝细胞胞浆内几乎无表达，而在模型组小鼠肝细胞胞浆内有显著表达（P0.05），与模型组相比，D4F组肝细胞胞浆内TNF-α表达明显减少（P0.05），较模型组减少42.4%；NF-κB在正常组小鼠肝细胞胞浆内有表达，在细胞核内几乎无表达，而在模型组小鼠肝细胞胞浆及胞核内均显著表达（P0.05），与模型组相比，D4F组肝细胞胞核内NF-κB表达明显减少（P0.05），为模型组的45.5%；sD4F组与模型组比较各项指标无统计学差异。 4.蛋白印迹结果显示：TNF-α在正常组小鼠肝组织内几乎无表达，而在模型组小鼠肝组织内有显著表达（P0.05），与模型组相比，D4F组肝组织内TNF-α表达明显减少（P0.05）；NF-κB在正常组小鼠肝组织内有少量表达，在细胞核内几乎无表达，而在模型组小鼠肝组织内均显著表达（P0.05），与模型组相比，D4F组肝组织内NF-κB表达明显减少（P0.05）；sD4F组与模型组比较各项指标无统计学差异。结论高脂饮食引起Apo E-/-小鼠肝脏脂质沉积，并继发肝脏炎性改变；ApoAI模拟肽D4F能够减少高脂饮食Apo E-/-小鼠肝脏脂质的沉积，同时能够减轻肝脏组织的炎性改变，说明D4F具有预防小鼠肝脏脂肪变性及炎性的作用；ApoAI模拟肽D4F对小鼠肝脏脂肪样变及炎性变化的预防效能可能涉及抑制NF-κB通路的表达以及减轻炎症因子TNF-α、IL-6的释放。
[Abstract]:objective
In this experiment, a mouse model of apolipoprotein E knockout (Apo E-/-), similar to human nonalcoholic fatty liver disease (NALFD), was fed with high fat feed, and the effect of apolipoprotein AI (ApoAI) analogue peptide D4F on lipid metabolism in liver of mice was observed. The dry pre preparation of Apo A mimic peptide D4F on liver adipose change and inflammation in mice was discussed. To further explore its possible mechanism of action, provide a basis for clinical treatment of NALFD.
Method
24 8 weeks male Apo E-/- mice were randomly divided into 3 groups (n=8) after two weeks of high fat (15.8%) and high cholesterol (1.25%). They were injected intraperitoneally with physiological saline (model group), disorder analogue peptide sD4F (1mg/kg. D, sD4F group) and Apo AI analog peptide D4F (1mg/kg D, group). The normal saline was executed as a normal control group after.6 weeks. ELISA method was used to detect the content of TNF- alpha and IL-6 in Apo E-/- and normal mice plasma. The pathological changes of liver tissue were observed by HE staining, and the degree of fatty degeneration in the liver group was evaluated by the method of oil red O staining, and the immunohistochemical method was used to detect Apo. E-/- and the expression and distribution of nuclear factor kappa B, TNF- alpha in liver of normal mice liver tissues, and the determination of NF- kappa B and TNF- alpha in liver tissues by Western Blotting. The experimental data were statistically analyzed by the SPSS13.0 statistical package. The two groups were compared with single factor variance, and the experimental data were expressed as mean standard deviation, P0.05 recognition. There was a statistical difference between the differences.
Result
1. the pathological sections of liver tissue in mice showed that a large number of lipid droplets were accumulated in the liver cells of the model group and the sD4F group, accompanied by different degrees of fatty degeneration and infiltration of interlobular inflammatory cells in different degrees, and the degree of fatty degeneration in the liver cells of the D4F group was significantly reduced and the interlobular inflammatory cells were soaked. There was no significant difference between the control group and the control group.
2. the test results of serum cytokines in each group showed that compared with the control group, the level of serum TNF- A and IL-6 increased significantly in the model group ApoE-/- mice, and was statistically significant (P0.05). Compared with the model mice, the content of TNF- alpha and IL-6 in the D4F group decreased significantly, which were respectively 48.6% and 63.4% of the model group, and the difference was statistically significant. Academic meaning (P0.05); there was no significant difference between sD4F group and model group.
3. the results of immunohistochemical staining showed that TNF- alpha was almost no expression in the cytoplasm of the normal mice liver cells, but in the cytoplasm of the mice in the model group (P0.05), the expression of TNF- alpha in the cytoplasm of hepatocytes in the D4F group decreased significantly (P0.05), which was less than that in the model group (P0.05), and NF- kappa B had a table in the cytoplasm of the normal mice liver cells. There was almost no expression in the nucleus, but in the cytoplasm and nucleus of the liver cells in the model group (P0.05). Compared with the model group, the expression of NF- kappa B in the cell nucleus of the D4F group was significantly decreased (P0.05), 45.5% in the model group, and in the sD4F group, there was no statistical difference between the model group and the model group.
The results of 4. protein blot showed that TNF- alpha was almost no expression in the liver tissues of normal mice, but in the liver tissues of the model mice (P0.05), the expression of TNF- alpha in the liver tissue of the D4F group was significantly decreased (P0.05), and the NF- kappa B had a small amount of expression in the normal mice liver tissue, and there was almost no expression in the nucleus of the normal group. The expression of NF- in the liver tissues of the model group was significantly (P0.05). Compared with the model group, the expression of NF- kappa B in the liver tissue of the D4F group was significantly decreased (P0.05), and there was no statistical difference between the sD4F group and the model group.
conclusion
High fat diet induced the liver lipid deposition in Apo E-/- mice and secondary liver inflammatory changes; ApoAI mimic peptide D4F can reduce the lipid deposition in the liver of Apo E-/- mice with high fat diet, and reduce the inflammatory changes in liver tissue, indicating that D4F has the effect on preventing the liver fatty degeneration and inflammation in mice; ApoAI analogue peptide D4F on mice liver The preventive efficacy of visceral lipoid and inflammatory changes may involve inhibiting the expression of NF- - B pathway and alleviating the release of inflammatory factor TNF- alpha and IL-6.
【学位授予单位】：泰山医学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R575.5

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