【摘要】：目的：建立高脂飼料喂養(yǎng)的非酒精性脂肪性肝�。∟onalcoholic fatty liverdisease，NAFLD）小鼠模型，動(dòng)態(tài)觀察鋅alpha2糖蛋白（Zinc alpha2glycoprotein,ZAG）在NAFLD發(fā)展中的表達(dá)變化，初步探討ZAG與NAFLD的關(guān)系。 方法：48只8周齡雄性C57BL/6小鼠隨機(jī)分成高脂飲食組(HFD)和標(biāo)準(zhǔn)飲食組(SD)，分別飼養(yǎng)4周、8周、12周和16周。HE染色分析肝臟組織病理學(xué)改變；ELISA法檢測(cè)血清ZAG以及炎癥因子TNF-α、IL-6、IL-8和IL-1β水平；新鮮肝臟勻漿檢測(cè)其甘油三酯（Triglyceride，，TG）含量；實(shí)時(shí)熒光定量PCR檢測(cè)肝臟ZAG的mRNA表達(dá)水平，Western-blotting技術(shù)及免疫組化法檢測(cè)肝臟ZAG的蛋白表達(dá)水平。 結(jié)果：4周開(kāi)始，HFD組小鼠血清TNF-α較SD組小鼠明顯升高(P0.05)，且隨NAFLD的發(fā)生而持續(xù)增加。8周起HFD組小鼠的IL-6、IL-8以及IL-1β逐漸升高(P0.05)。4周起，HFD組小鼠肝臟ZAG的mRNA及蛋白水平逐漸下降(P0.05)，而血清ZAG濃度從8周開(kāi)始明顯降低(P0.05)。 結(jié)論：高脂飲食誘導(dǎo)C57BL/6小鼠發(fā)生NAFLD，其機(jī)制可能與肝臟ZAG表達(dá)水平的降低有關(guān)。 目的：建立MCD飼料喂養(yǎng)的非酒精性脂肪性肝炎（Nonalcoholic steatohepatitis，NASH）小鼠模型，尾靜脈注射rAAV2-ZAG-CMV-EGFP誘導(dǎo)其肝臟ZAG過(guò)表達(dá)，探討ZAG在NAFLD小鼠肝臟脂質(zhì)代謝及炎癥反應(yīng)中的作用。 方法：54只8周齡雄性C57BL/6小鼠適應(yīng)性喂養(yǎng)1周，按體重隨機(jī)分為Control組（12只）、MCD+PBS組（14只）、MCD+ZAG組（14只）以及MCD+GFP組（14只）。 Control組小鼠給予標(biāo)準(zhǔn)飼料喂養(yǎng)8周，其余3組首先給予MCD飼料喂養(yǎng)4周，MCD+ZAG組小鼠給予尾靜脈注射rAAV2-ZAG-CMV-EGFP2×1011vg/只,MCD+GFP組給予同劑量rAAV2-CMV-EGFP，MCD+PBS組給予同體積的PBS，繼續(xù)MCD飼料喂養(yǎng)4周。8周末處死所有小鼠，收集血清和肝臟組織。HE及油紅O染色觀察肝臟組織病理學(xué)改變；生化及ELISA法檢測(cè)空腹血糖、血脂、炎癥因子TNF-α、IL-6、IL-8、IL-1β水平以及肝臟TG含量；RT-qPCR、Western-blotting及免疫組化分別檢測(cè)肝臟ZAG、脂代謝相關(guān)基因以及炎癥因子的mRNA和蛋白水平表達(dá)。 結(jié)果：8周末，MCD飼料喂養(yǎng)的小鼠，其飲食量、體重、肝濕重、血清TG、TC、LDL、HDL、FPG明顯降低（P0.05），肝臟指數(shù)、ALT、AST、血清TNF-α、IL-6、IL-8、IL-1β以及肝臟TG含量明顯升高（P0.05），肝臟脂肪分解相關(guān)基因HSL、PPARα、CPT1A的mRNA和蛋白表達(dá)水平明顯降低（P0.05），肝臟相關(guān)炎癥因子TNF-α、IL-1β、ICAM-1、MCP-1、CD68、F4/80的mRNA和蛋白表達(dá)增加（P0.05），同時(shí)，IL-6、IL-8的蛋白表達(dá)水平也明顯增加（P0.05）。ZAG過(guò)表達(dá)的NASH小鼠，其肝濕重、血清TG、TC、肝臟指數(shù)、ALT、AST、血清TNF-α、IL-6、IL-1β水平也有所降低（P0.05），血清LDL、HDL、FPG、IL-8無(wú)明顯差異（P0.05）。ZAG過(guò)表達(dá)組小鼠，其肝臟脂肪分解相關(guān)基因HSL、PPARα、CPT1A的mRNA、蛋白水平增加（P0.05），而炎癥因子TNF-α、IL-1β、ICAM-1、MCP-1、CD68、F4/80的mRNA和蛋白表達(dá)水平降低（P0.05），且IL-6、IL-8的蛋白水平也明顯降低（P0.05）。 結(jié)論：ZAG能夠改善MCD飼料誘導(dǎo)的NAFLD小鼠肝臟脂肪變及炎癥反應(yīng)，可能與ZAG調(diào)節(jié)肝臟脂代謝相關(guān)基因的表達(dá)以及抑制炎癥因子的表達(dá)有關(guān)。
[Abstract]:AIM: To establish a mouse model of non-alcoholic fatty liver disease (NAFLD) fed with high-fat diet and observe the expression of Zinc alpha2 glycoprotein (ZAG) in the development of NAFLD.
Methods: 48 8-week-old male C57BL/6 mice were randomly divided into high-fat diet group (HFD) and standard diet group (SD) and fed for 4 weeks, 8 weeks, 12 weeks and 16 weeks respectively. The expression of ZAG mRNA in liver was detected by real-time fluorescence quantitative PCR, and the expression of ZAG protein in liver was detected by Western-blotting and immunohistochemistry.
Results: The serum TNF-alpha level of HFD mice was significantly higher than that of SD mice (P 0.05), and increased with the occurrence of NAFLD. The levels of IL-6, IL-8 and IL-1 beta in HFD mice increased gradually (P 0.05).
CONCLUSION: High fat diet induces NAFLD in C57BL/6 mice, which may be related to the decrease of ZAG expression in liver.
AIM: To establish a non-alcoholic steatohepatitis (NASH) mouse model fed with MCD diet and to investigate the role of ZAG in lipid metabolism and inflammation of NAFLD mice by tail vein injection of rAAV2-ZAG-CMV-EGFP.
Methods: Fifty-four eight-week-old male C57BL/6 mice were randomly divided into control group (12 mice), MCD+PBS group (14 mice), MCD+ZAG group (14 mice) and MCD+GFP group (14 mice). Control group was fed with standard diet for 8 weeks. The other three groups were fed with MCD for 4 weeks. MCD+ZAG group was given rAAV2-ZAG-CMV-EG by tail vein injection. FP2 *1011vg/mouse, MCD+GFP group was given the same dose of rAAV2-CMV-EGFP, MCD+PBS group was given the same volume of PBS, MCD+PBS group was fed for 4 weeks. At the end of 8 weeks, all the mice were killed and serum and liver tissues were collected. The expression of ZAG, lipid metabolism related genes and inflammatory factors mRNA and protein were detected by RT-qPCR, Western-blotting and immunohistochemistry.
Results: At the end of 8 weeks, the diet, body weight, liver wet weight, serum TG, TC, LDL, HDL, FPG decreased significantly (P 0.05), liver index, ALT, AST, serum TNF-a, IL-6, IL-8, IL-1 beta and liver TG content increased significantly (P 0.05), hepatic fat decomposition related genes HSL, PPARa, CPT1A mRNA and protein expression levels decreased significantly (P 0.05). The mRNA and protein expression of liver-related inflammatory factors TNF-a, IL-1beta, ICAM-1, MCP-1, CD68, F4/80 were increased (P 0.05). At the same time, the protein expression of IL-6 and IL-8 was also significantly increased (P 0.05). The liver wet weight, serum TG, TC, liver index, ALT, AST, serum TNF-a, IL-6, IL-1beta levels were also decreased in NASH mice with ZAG overexpression (P 0.05). There was no significant difference between ZAG overexpression group and control group (P 0.05). The mRNA and protein levels of hepatic fat-decomposition-related genes HSL, PPAR-a, CPT1A increased (P 0.05), while the mRNA and protein levels of inflammatory factors TNF-a, IL-1beta, ICAM-1, MCP-1, CD68, F4/80 decreased (P 0.05), and the protein levels of IL-6 and IL-8 decreased significantly (P 0.05).
CONCLUSION: ZAG can improve hepatic steatosis and inflammation induced by MCD in NAFLD mice, which may be related to ZAG regulating the expression of lipid metabolism related genes and inhibiting the expression of inflammatory factors.
【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】：2014
【分類(lèi)號(hào)】：R575.5

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