本文選題：非酒精性脂肪性肝病 + 腫瘤壞死因子-α　； 參考：《泰山醫(yī)學院》2014年碩士論文

【摘要】：目的 本實驗通過高脂飼料喂養(yǎng)建立與人類非酒精性脂肪性肝�。∟ALFD）發(fā)病過程相似的載脂蛋白E基因敲除（Apo E-/-）小鼠模型，觀察載脂蛋白AI（ApoAI）模擬肽D4F對小鼠肝臟脂質代謝的影響，探討Apo A模擬肽D4F對小鼠肝臟脂肪樣變及炎性的干預作用并進一步探討其可能的作用機制，，為臨床上對NALFD的藥物治療提供依據(jù)。 方法 24只8周齡雄性Apo E-/-小鼠，經(jīng)過兩周高脂（15.8%）與高膽固醇（1.25%）喂食后隨機分為3組(n=8），分別腹腔注射生理鹽水(模型組）、紊亂模擬肽sD4F (1mg/kg·d，sD4F組）和Apo AI模擬肽D4F（1mg/kg·d，D4F組）。同周齡雄性C57BL/6J小鼠喂食普通飲食并給予腹腔注射生理鹽水作為正常對照組。6周后處死，ELISA法檢測Apo E-/-及普通小鼠血漿中炎癥因子TNF-α、IL-6的含量，并取小鼠肝臟組織，采用HE染色觀察其病理形態(tài)的改變，同樣采用油紅O染色方法評估肝組織脂肪變性程度，免疫組織化學方法檢測Apo E-/-及普通小鼠肝組織核因子-κB、TNF-α在肝臟中的表達與分布，同時采用Western Blotting方法檢測肝組織中NF-κB、TNF-α的含量。實驗數(shù)據(jù)采用SPSS13.0統(tǒng)計軟件包進行統(tǒng)計，兩組間比較采用單因素方差檢驗，實驗數(shù)據(jù)以均數(shù)±標準差表示，P0.05認為差異有統(tǒng)計學差異。 結果 1.小鼠肝臟組織病理切片結果顯示：光學顯微鏡下可見模型組及sD4F組小鼠的肝臟細胞中出現(xiàn)大量的脂質滴的累積，并伴有不同程度脂肪變性及小葉間炎性細胞的浸潤；D4F組小鼠的肝臟細胞脂肪變性程度顯著減輕，小葉間炎性細胞浸潤明顯減少，與對照組小鼠相比無明顯差別。 2.對各組小鼠的血清細胞因子的檢測結果顯示：與對照組小鼠比較，模型組ApoE-/-小鼠血清TNF-α、IL-6的水平增高顯著，并有統(tǒng)計學意義（P0.05）；與模型組小鼠相比，D4F組血清TNF-α、IL-6的含量明顯下降，分別為模型組的48.6%和63.4%，差異有統(tǒng)計學意義（P0.05）；sD4F組與模型組比較各項指標無統(tǒng)計學差異。 3.免疫組化結果顯示：TNF-α在正常組小鼠肝細胞胞漿內(nèi)幾乎無表達，而在模型組小鼠肝細胞胞漿內(nèi)有顯著表達（P0.05），與模型組相比，D4F組肝細胞胞漿內(nèi)TNF-α表達明顯減少（P0.05），較模型組減少42.4%；NF-κB在正常組小鼠肝細胞胞漿內(nèi)有表達，在細胞核內(nèi)幾乎無表達，而在模型組小鼠肝細胞胞漿及胞核內(nèi)均顯著表達（P0.05），與模型組相比，D4F組肝細胞胞核內(nèi)NF-κB表達明顯減少（P0.05），為模型組的45.5%；sD4F組與模型組比較各項指標無統(tǒng)計學差異。 4.蛋白印跡結果顯示：TNF-α在正常組小鼠肝組織內(nèi)幾乎無表達，而在模型組小鼠肝組織內(nèi)有顯著表達（P0.05），與模型組相比，D4F組肝組織內(nèi)TNF-α表達明顯減少（P0.05）；NF-κB在正常組小鼠肝組織內(nèi)有少量表達，在細胞核內(nèi)幾乎無表達，而在模型組小鼠肝組織內(nèi)均顯著表達（P0.05），與模型組相比，D4F組肝組織內(nèi)NF-κB表達明顯減少（P0.05）；sD4F組與模型組比較各項指標無統(tǒng)計學差異。 結論 高脂飲食引起Apo E-/-小鼠肝臟脂質沉積，并繼發(fā)肝臟炎性改變；ApoAI模擬肽D4F能夠減少高脂飲食Apo E-/-小鼠肝臟脂質的沉積，同時能夠減輕肝臟組織的炎性改變，說明D4F具有預防小鼠肝臟脂肪變性及炎性的作用；ApoAI模擬肽D4F對小鼠肝臟脂肪樣變及炎性變化的預防效能可能涉及抑制NF-κB通路的表達以及減輕炎癥因子TNF-α、IL-6的釋放。
[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.
【學位授予單位】：泰山醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R575.5

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