【摘要】：目的:果糖攝入增多可引起肝臟內(nèi)脂質(zhì)蓄積,進(jìn)而導(dǎo)致肝臟發(fā)生脂肪變性。有研究發(fā)現(xiàn)ω-3多不飽和脂肪酸(如DHA)對(duì)肝臟的脂肪變性有一定的保護(hù)作用。本研究擬以原代肝臟細(xì)胞體外培養(yǎng)為研究體系,探討DHA是否能夠改善果糖誘導(dǎo)的肝臟脂質(zhì)蓄積及可能的分子機(jī)制。方法:選用正常雄性的C57 BL/6J小鼠,采用肝臟肝門靜脈灌流的方法,獲得肝臟細(xì)胞;得到的肝臟細(xì)胞分別用果糖、果糖和DHA、DHA、PBA和果糖、TM處理細(xì)胞24小時(shí)以后進(jìn)行油紅O染色觀察細(xì)胞內(nèi)紅染脂滴蓄積的情況;Real-time PCR檢測肝細(xì)胞內(nèi)目標(biāo)基因表達(dá)水平;Western blotting檢測肝細(xì)胞內(nèi)ACC、SREBP、ACOX1、GRP78/BIP、IRE、p-IRE蛋白表達(dá)水平。結(jié)果:油紅O染色結(jié)果表明原代肝細(xì)胞在果糖和衣霉素作用下發(fā)生明顯的脂質(zhì)蓄積,在DHA的作用下細(xì)胞內(nèi)脂質(zhì)蓄積明顯減少,而果糖和DHA復(fù)合處理組細(xì)胞內(nèi)脂質(zhì)蓄積也明顯減輕,同時(shí)PBA預(yù)處理后再加入果糖干預(yù)肝細(xì)胞脂質(zhì)蓄積明顯減輕;Real-time PCR結(jié)果顯示原代肝細(xì)胞在果糖和衣霉素作用下FAS、ACC、SREBP、GRP78的mRNA表達(dá)水平較對(duì)照組明顯上調(diào);DHA處理組FAS、ACC、SREBP、GRP78的mRNA表達(dá)水平較對(duì)照組無明顯變化,CPT1α、ACOX1明顯上調(diào);果糖和DHA復(fù)合處理組FAS、ACC、GRP78的mRNA表達(dá)水平較果糖組明顯下調(diào),CPT1α、ACOX1明顯上調(diào);果糖和PBA復(fù)合處理組FAS、ACC、SREBP、GRP78的mRNA表達(dá)水平較果糖組明顯下調(diào),CPT1α、ACOX1無明顯變化。Western blotting結(jié)果提示原代肝細(xì)胞在果糖和衣霉素作用下ACC、SREBP、GRP78/BIP、IRE和p-IRE蛋白表達(dá)較對(duì)照組顯著增強(qiáng);DHA處理組ACC、SREBP、GRP78/BIP、IRE和p-IRE蛋白表達(dá)較對(duì)照組無明顯變化,ACOX1蛋白表達(dá)明顯增強(qiáng);果糖和DHA復(fù)合處理組與果糖和PBA復(fù)合處理組ACC、SREBP、GRP78/BIP、IRE和p-IRE蛋白表達(dá)較果糖組蛋白表達(dá)明顯減弱,ACOX1蛋白表達(dá)明顯增強(qiáng)。結(jié)論:果糖可通過誘導(dǎo)內(nèi)質(zhì)網(wǎng)應(yīng)激激活脂質(zhì)代謝相關(guān)基因的表達(dá)進(jìn)而導(dǎo)致肝臟脂肪變性,DHA通過抑制內(nèi)質(zhì)網(wǎng)應(yīng)激相關(guān)基因的表達(dá)進(jìn)而緩解果糖誘導(dǎo)的肝臟脂肪變性。飲食中添加少量的DHA可以預(yù)防果糖攝入過多引發(fā)的非酒精行脂肪肝的發(fā)生,從而減少疾病的發(fā)生。
[Abstract]:Objective: increased fructose intake may lead to lipid accumulation in the liver, which leads to fatty degeneration of the liver. It has been found that 蠅 -3 polyunsaturated fatty acids (such as DHA) have a protective effect on liver steatosis. The aim of this study was to investigate whether DHA could improve hepatic lipid accumulation induced by fructose and its possible molecular mechanism with primary hepatocyte culture in vitro. Methods: normal male C57 BL/6J mice were used to obtain liver cells by hepatic portal vein perfusion, and the liver cells were obtained by fructose. After treated with fructose and DHADHAPBA and fructose TM for 24 hours, oil red O staining was performed to observe the accumulation of lipid droplets in the cells. Real-time PCR was used to detect the expression level of target genes in hepatocytes. Western blotting was used to detect the expression level of ACC-SREBPN-GRP78 / BIPIREp-IRE in hepatocytes. Results: the results of oil red O staining showed that the primary hepatocytes had obvious lipid accumulation under the action of fructose and itlamycin, and the intracellular lipid accumulation was obviously decreased under the action of DHA. The intracellular lipid accumulation in fructose and DHA treatment group was also significantly reduced. At the same time, after pretreatment with PBA, fructose was added to inhibit lipid accumulation of hepatocytes significantly. The results showed that the expression of mRNA in primary hepatocytes was significantly up-regulated in the primary hepatocytes treated with fructose and chlortetracycline as compared with the control group. The mRNA expression of FASACC-SREBPnGRP78 was up-regulated by fructose and chlortetracycline. Compared with the control group, the level of CPT1 偽 and ACOX1 was up-regulated. Compared with fructose and DHA group, the expression of mRNA in FASA ACC-GRP78 was significantly down-regulated in fructose group, and the expression of CPT1 偽 and ACOX1 was significantly up-regulated in fructose group. Compared with fructose group, fructose and PBA combined treatment group had significantly down-regulated the expression of mRNA in FASACC-SREBPnGRP78. Western blotting showed that the expression of ACC-SREBPnGRP78 / BIPIIRE and p-IRE protein in primary hepatocytes treated with fructose and chlortetracycline was significantly enhanced than that in control group. Compared with the control group, the expression of ACOX1 protein increased significantly in the expression of ACC-SREBPnGRP78 / BIPP78 and p-IRE protein. Compared with fructose and DHA groups and fructose and PBA groups, the expression of ACC-SREBPG-GRP78 / BIPII-IRE and p-IRE protein was significantly decreased compared with fructose histone group and fructose combined treatment group and fructose and PBA combined treatment group, and the expression of ACOX1 protein was significantly increased. Conclusion: fructose can induce endoplasmic reticulum stress to activate the expression of genes related to lipid metabolism, and then induce hepatic steatosis by inhibiting the expression of endoplasmic reticulum stress-related genes, thereby alleviating fructose induced hepatic steatosis. Dietary supplementation of a small amount of DHA can prevent the occurrence of nonalcoholic fatty liver caused by excessive fructose intake, thus reducing the incidence of disease.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R575

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