【摘要】：目的：非酒精性脂肪性肝病(non-alcoholic fatty liver diseases，NAFLD)是臨床上最為常見的的慢性肝病之一，其炎性反應(yīng)貫穿了整個病變過程。近年來發(fā)現(xiàn)ω-3系多不飽和脂肪酸(ω-3polyunsaturated fatty acid，ω-3PUFA)對NAFLD炎性損傷有正向干預(yù)作用，但具體作用機制尚不明確，所以本課題旨在初步探討ω-3PUFA緩解NAFLD炎性損傷的可能作用機制。 方法：采用高脂飼喂建立NAFLD動物模型。選用體重160~180g的雄性SD大鼠，隨機分為4組，每組48只：正常飼料飼喂的對照組（CON組）、高脂（20%豬油）飼喂的NAFLD模型組（HFD組）、高脂飼料基礎(chǔ)上加魚油（10%豬油+10%魚油）的魚油干預(yù)組（FOH組）、高脂飼料基礎(chǔ)上加紫蘇油(14.83%豬油+5.17%紫蘇油)干預(yù)的紫蘇油干預(yù)組（POH組），分別于實驗的4w、8w、12w和16w時間點斷頭取血處死（每時間點12只/組），稱取體重、肝重，計算肝指數(shù)。采用酶法檢測血清肝功（ALT、AST）、血脂（TCTG、HDL-C、LDL-C）、SOD、GST和FFA變化情況；HE染色觀察肝細胞氣球樣變、脂肪變性程度和炎性反應(yīng)程度；Masson染色觀察肝組織纖維化分期；免疫組織化學技術(shù)檢測巨噬細胞特異性抗體CD68蛋白的表達；使用熒光實時定量PCR技術(shù)檢測肝組織中IL-6、IL-1β、TNF-、MCP-1、NF-κB P65的mRNA的表達水平。 結(jié)果：（1）HFD組較CON組8w時大鼠肝指數(shù)明顯升高，肝細胞氣球樣變明顯，呈現(xiàn)出大小泡混合型肝細胞脂肪變性，小葉內(nèi)和匯管區(qū)炎性細胞浸潤，纖維化明顯，即發(fā)生了典型的NASH病變。另外，8w時血清ALT、TCH、FFA水平均顯著性升高，HDL-C顯著性降低。至12w時AST、TG、LDL-C也顯著性升高，SOD顯著性降低，16w時GST顯著性升高。炎性細胞因子（IL-6、IL-1β、TNF-）、趨化因子MCP-1的mRNA表達均在8w都顯著性升高，到12w時NF-κB P65的mRNA表達也顯著性升高。蛋白水平，16w時CD68陽性表達明顯增多，巨噬細胞大片聚集。 （2）魚油干預(yù)后，肝細胞氣球樣變，脂肪變性，小葉內(nèi)和匯管區(qū)炎性細胞浸潤，纖維化均有不同程度的改善。并且4w時血清ALT、TCH、FFA較HFD組顯著性降低；到8w時LDL-C、GST較HFD組顯著性降低；12w時AST、TG較HFD組顯著性降低，SOD較HFD組顯著性升高。RNA水平上，炎性細胞因子IL-6在4w時開始顯著性降低，TNF-在8w時開始顯著性降低，IL-1β在12w時開始顯著性降低；趨化因子MCP-1在8w時開始顯著性降低；NF-κB P65在12w時開始顯著性降低。蛋白水平，16w時CD68陽性表達明顯減少。 （3）紫蘇油干預(yù)后，肝細胞氣球樣變，脂肪變性，小葉內(nèi)和匯管區(qū)炎性細胞浸潤，纖維化也呈現(xiàn)不同程度的改善。4w時血清ALT較HFD組顯著性降低；到8w時TCH、LDL-C、FFA、GST較HFD組顯著性降低；12w時AST、TG較HFD組顯著性降低，SOD較HFD組顯著性升高。RNA水平，炎性細胞因子IL-6有降低但沒有顯著性變化， TNF-、IL-1β在12w時開始顯著性降低；趨化因子MCP-1在12w時開始顯著性降低；NF-κB P65在16w時開始顯著性降低。蛋白水平，16w時CD68陽性表達明顯減少。 結(jié)論：(1)高脂飲食8w時，大鼠肝臟進展到了NASH病程，魚油、紫蘇油干預(yù)不同程度緩解了NAFLD炎性損傷。 (2) ω-3PUFA可能通過NF-κB信號通路，抑制炎性細胞因子的產(chǎn)生，，最終緩解了NAFLD炎性進程。
[Abstract]:Objective: Non-alcoholic fatty liver disease (NAFLD) is one of the most common chronic liver diseases. In recent years, it has been found that the trans-3 PUFA has a positive effect on the inflammatory injury of NAFLD, but the mechanism of specific action is not clear. Methods: The animal model of NAFLD was established by high-fat feeding. Type: Male SD rats weighing 160-180 g were randomly divided into 4 groups,48 rats in each group: control group (CON group) fed with normal feed, and NAFLD model group (HFD group) fed with high fat (20% lard). (10% lard + 10% fish oil) on the basis of high-fat diet (FOH group), and perilla oil (14.83% lard + 5.17% perilla oil) was added to the base of high-fat diet to intervene with Perilla oil intervention group (POH group). At the time point of w, 12w, and 16w, the blood was killed (12/ group at each time point), and the weight and liver weight were weighed, and the liver finger was calculated. The changes of serum liver function (ALT, AST), blood lipid (TCTG, HDL-C, LDL-C), SOD, GST and FFA in serum were detected by enzyme method. The expression of IL-6, IL-1, TNF-, MCP-1, NF-CDB P65 in liver tissue was detected by fluorescent real-time quantitative PCR (RT-PCR). Results: (1) The liver index of the rats was significantly higher in the HFD group than that of the CON group, and the balloon of the liver cells was obvious, presenting a large-size mixed type of hepatocyte fat degeneration, infiltration of the inflammatory cells in the lobular and foreign exchange tubes, and the obvious fibrosis, that is, the typical NAS H. In addition, the levels of ALT, TCH and FFA in serum increased significantly at 8w, and HDL-C was significant. The results showed that AST, TG, LDL-C in AST, TG and LDL-C increased significantly at 12w. The expression of IL-6, IL-1, TNF-and the expression of MCP-1 in the expression of MCP-1 increased significantly at 8w. The expression of CD68 was significantly increased at the level of protein and 16w, and the macrophage was large. (2) After the intervention of fish oil, the cell infiltration and fibrosis of the liver cells were different after the intervention of the fish oil. The levels of serum ALT, TCH and FFA were significantly lower than those in the HFD group at the time of 4w. The levels of LDL-C and GST were significantly lower at 8w, and the levels of AST and TG in the lower group were significantly lower than those in the HFD group, and the SOD was lower than that of the HFD group. There was a significant increase in the level of the RNA, the level of IL-6 of the inflammatory cytokines decreased significantly at 4w, the level of TNF-1 was significantly lower at 8w, the level of IL-1 was significantly lower at 12 w, and the expression of the chemokine MCP-1 was significantly lower at 8w; and the NF-EMAB P65 started at 12w Significant decrease. Protein level, CD68 positive table at 16 w (3) After the intervention of Perilla oil, the cell infiltration and fibrosis of the liver cell balloon and the inflammatory cells of the interlobular area and the sink area were improved, and the level of the serum ALT was significantly decreased at the time of 4w. The levels of TCH, LDL-C, FFA and GST in the group were lower than those of the HFD group at the time of 8w. At 12w AST, TG was significantly lower than that in the HFD group, and the SOD was higher than that of the HFD group. There was a significant increase in the level of the D group. The level of the RNA, the IL-6 of the inflammatory cytokine, but no significant change, the TNF-, IL-1 level began to decrease significantly at 12w; the chemokines MCP-1 began to significantly decrease at 12w; and the NF-EMAB P65 at 16 w Start significant reduction. Protein level, CD68 male at 16 w Conclusion: (1) When the high-fat diet is 8w, the liver of the rat has progressed to the course of NASH, fish oil and Perilla oil. NAFLD inflammatory injury. (2) The 3-3 PUFA may pass through the NF-B signal pathway to inhibit the production of inflammatory cytokines and eventually slow down
【學位授予單位】：武漢輕工大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R575.5

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