【摘要】：非酒精性脂肪肝是高發(fā)且嚴(yán)重危害人類健康的飲食相關(guān)性肝損傷，從膳食角度尋找其防治因子具有重要意義。前期研究表明萊菔硫烷（SFN）可減輕酒精性肝損傷及調(diào)控脂代謝，但對(duì)非酒精性肝損傷的作用及機(jī)制尚無深入探討。本研究探討SFN預(yù)保護(hù)人肝細(xì)胞HHL-5后，能否抵抗油酸/軟脂酸對(duì)肝細(xì)胞脂代謝的病理性損傷，重點(diǎn)探究內(nèi)質(zhì)網(wǎng)應(yīng)激調(diào)控通路的作用，主要研究結(jié)果如下。 油酸/軟脂酸（1:2）作誘導(dǎo)劑，100-500μmol/L作用于HHL-5細(xì)胞，油紅O染色檢測(cè)脂滴，再檢測(cè)肝細(xì)胞代謝酶和甘油三酯（TC）及膽固醇（TG），發(fā)現(xiàn)油酸/軟脂酸300μmol/L時(shí)，多數(shù)細(xì)胞內(nèi)出現(xiàn)紅色圓珠狀且非常飽滿的脂滴，誘導(dǎo)劑劑量增大，脂滴增多。油酸/軟脂酸（1:2）300μmol/L作用24h，TC和TG含量明顯升高（P0.01），谷丙轉(zhuǎn)氨酶（ALT）與谷草轉(zhuǎn)氨酶（AST）活性顯著升高；誘導(dǎo)劑濃度大于400μmol/L，細(xì)胞死亡。因此，將油酸/軟脂酸（1:2）300μmol/L作用24h作為誘導(dǎo)肝細(xì)胞HHL-5脂代謝異常的作用方式。SFN分別以時(shí)間效應(yīng)和劑量效應(yīng)方式預(yù)保護(hù)細(xì)胞，再用油酸/軟脂酸刺激24h，發(fā)現(xiàn)TG和TC含量與陽性對(duì)照組比較顯著降低（P0.01），10μmol/L SFN對(duì)TG與TC含量降低最為明顯，抑制AST和ALT增加，抑制后水平與陰性對(duì)照組接近。 以內(nèi)質(zhì)網(wǎng)為切入點(diǎn)，透射電鏡檢測(cè)結(jié)果表明，油酸/軟脂酸作用細(xì)胞24h可引起內(nèi)質(zhì)網(wǎng)擴(kuò)張，提示內(nèi)質(zhì)網(wǎng)功能受損，同時(shí)影響線粒體功能，表現(xiàn)為線粒體顏色加深。SFN預(yù)保護(hù)后能明顯改善內(nèi)質(zhì)網(wǎng)的擴(kuò)張，內(nèi)質(zhì)網(wǎng)結(jié)構(gòu)基本恢復(fù)到陰性對(duì)照組水平，表現(xiàn)為內(nèi)質(zhì)網(wǎng)成條索狀，，并對(duì)線粒體損傷明顯改善。Real-timePCR檢測(cè)內(nèi)質(zhì)網(wǎng)調(diào)控蛋白XBP1、GRP78轉(zhuǎn)錄水平的表達(dá)情況。發(fā)現(xiàn)油酸/軟脂酸（1:2）300μmol/L誘導(dǎo)肝細(xì)胞24h后，XBP1與GRP78mRNA表達(dá)量顯著增加。10μmol/L SFN使二者表達(dá)量比誘導(dǎo)組降低。SFN作用時(shí)間延長，XBP1與GRP78mRNA表達(dá)量減小，24h最顯著。流式細(xì)胞術(shù)檢測(cè)內(nèi)質(zhì)網(wǎng)調(diào)控網(wǎng)絡(luò)中心調(diào)控因子Ca2+，發(fā)現(xiàn)油酸/軟脂酸能誘導(dǎo)內(nèi)質(zhì)網(wǎng)中Ca2+流出，胞漿中Ca2+濃度升高明顯高于陰性對(duì)照組。隨著SFN作用濃度增加，胞漿中鈣離子濃度下降。 本研究結(jié)果表明，SFN能明顯改善油酸/軟脂酸引起的肝細(xì)胞脂代謝異常，內(nèi)質(zhì)網(wǎng)調(diào)控在其中發(fā)揮重要作用。本研究將為SFN的開發(fā)應(yīng)用提供理論基礎(chǔ)。
[Abstract]:Non-alcoholic fatty liver is a kind of diet-related liver injury with high incidence and serious harm to human health. It is of great significance to look for its preventive and therapeutic factors from the point of view of diet. Previous studies have shown that sulforaphane (SFN) can attenuate alcoholic liver injury and regulate lipid metabolism, but the role and mechanism of sulforaphane (SFN) in non-alcoholic liver injury have not been thoroughly discussed. The aim of this study was to investigate whether SFN could protect human hepatocytes from the pathological damage of lipid metabolism induced by oleic acid / palmitate, and to explore the role of endoplasmic reticulum stress regulation pathway. The main results were as follows. Oleic acid / palmitic acid (1:2) acted on HHL-5 cells with 100-500 渭 mol/L, oil red O staining was used to detect lipid droplets, and liver cell metabolic enzymes, triglyceride (TC) and cholesterol (TG), were detected when oleic acid / palmitic acid was found to be 300 渭 mol/L. Red globular and very full lipid droplets were found in most cells. The dose of inducer increased and lipid droplets increased. The contents of TC and TG increased significantly (P0.01), the activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) increased significantly after treatment with oleic acid / palmitic acid (1:2) 300 渭 mol/L for 24 h, and the concentration of inducer was more than 400 渭 mol / L, the cell died. Therefore, 渭 mol/L of oleic acid / palmitate (1:2) was used to induce abnormal lipid metabolism of HHL-5 in hepatocytes for 24 hours. SFN preprotected the cells by time effect and dose effect, respectively. After stimulation with oleic acid / palmitic acid for 24 h, it was found that TG and TC decreased significantly (P0.01) 10 渭 mol/L SFN compared with that of the positive control group, the decrease of TG and TC content was the most obvious, the increase of AST and ALT was inhibited, and the level after inhibition was close to that of the negative control group. Using endoplasmic reticulum as the starting point, the results of transmission electron microscopy showed that oleic acid / palmitic acid could induce endoplasmic reticulum dilatation for 24 hours, suggesting that endoplasmic reticulum function was damaged and mitochondria function was affected. The results showed that the extension of endoplasmic reticulum (ER) could be improved obviously after pre-protection of mitochondria. The structure of endoplasmic reticulum (ER) returned to the level of negative control group, showing that the endoplasmic reticulum (ER) was striped. The expression of endoplasmic reticulum regulatory protein XBP1 GRP78 was improved by real-time PCR. It was found that the expression of XBP1 and GRP78mRNA in hepatocytes induced by oleic acid / palmitate (1:2) at 300 渭 mol/L for 24 hours increased significantly. 10 渭 mol/L SFN significantly decreased the expression of XBP1 and GRP78mRNA in hepatocytes. Flow cytometry was used to detect the central regulator of endoplasmic reticulum network (Ca2). It was found that oleic acid / palmitate could induce Ca2 outflow from the endoplasmic reticulum and the concentration of Ca2 in the cytoplasm was significantly higher than that in the negative control group. With the increase of the concentration of SFN, the concentration of Ca ~ (2 +) in the cytoplasm decreased. The results showed that SFN could significantly improve lipid metabolism of hepatocytes induced by oleic acid / palmitate, and endoplasmic reticulum regulation played an important role in it. This study will provide a theoretical basis for the development and application of SFN.
【學(xué)位授予單位】：哈爾濱工業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R575.5

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