三羟异黄酮对脂质代谢调控的研究

发布时间：2018-05-13 13:02

本文选题：三羟异黄酮 + PPARs　；参考：《合肥工业大学》2017年硕士论文

【摘要】：肝脏是脂类代谢的主要器官和重要场所,脂肪肝是常见的肝脏疾病之一,形成原因主要是肝细胞内脂肪储积积累过多、细胞脂肪化严重,导致脂肪代谢发生紊乱形成。有人已经检测出大豆异黄酮对实验性大鼠脂肪肝有显著的治疗作用。大豆基异黄酮是从豆科植物根部分离得到的一种天然次级代谢产物,它具有显著的抗癌、抑菌、抗氧化等作用。三羟异黄酮(Genistein,Gen)在大豆异黄酮活性功能中发挥重要作用,研究发现三羟异黄酮在脂质代谢过程中主要通过PPARs过氧化物酶体增殖物激活受体调控以及对代谢酶的表达与活性的调控发挥重要生理功能。目的:本文主要通过细胞体外培养试验来探索三羟异黄酮调控脂质代谢的途径,研究Gen对过氧化物酶体增殖物激活受体(PPARs)、肝X受体(LXR)、脂肪甘油三酯脂酶(Adipose Triglyceride lipase,ATGL)、激素敏感性甘油三酯脂肪酶(Hormone-sensitive lipase,HSL)调控表达中的规律,比较三羟异黄酮与PPAR激动剂对细胞活性的影响,以及探究Gen对脂肪化细胞中人肿瘤坏死因子TNF-α、瘦素Leptin的调控规律,为三羟异黄酮在营养保健、临床药理、调脂保肝等方面提供理论基础和试验依据。方法:实验对象为体外培养细胞模型,培养人体正常肝细胞L-02、人肝癌细胞HepG2、Hep3b以及小鼠前脂肪细胞3T3-L1四种细胞,将细胞分为空白组、模型组、Gen试剂组以及PPAR激动剂组。首先用MTT测定细胞在不同油酸(OA)浓度下的细胞活性,确定最佳的OA诱导浓度,建立细胞脂肪化模型。用Annexin V/PI双染色法对细胞进行染色处理,流式细胞仪检测细胞的凋亡情况;进一步验证Gen与PPAR激动剂及抑制剂比较对细胞的毒性影响;利用Gen、PPAR激活剂及抑制剂处理四种脂肪化细胞,实时荧光定量PCR测定细胞内ATGL、HSL、PPARα、PPARγ以及肝X受体LXR的mRNA表达量;酶联免疫法检测Gen、PPAR激活剂及抑制剂处理后的四种脂变细胞培养液中的TNF-α、Leptin含量变化。结果:(1)MTT细胞活力测定实验中,当油酸浓度高于1mM时,细胞活力大幅度降低且部分细胞漂浮坏死,细胞内TG含量显著性累积,当油酸浓度低于1mM时,细胞活力相近,细胞形态变化不明显。(2)流式细胞仪检测结果显示,三羟异黄酮对细胞脂肪化有保护作用,能提高细胞的活性。(3)Gen可以上调PPARs以及LXRα的mRNA表达量,其中对PPARs调控较LXRα显著,同时细胞中HSL和ATGL的m RNA表达量也有相应的提高。(4)Gen可以呈剂量效应调控TNF-α、Leptin浓度。结论:使用1mM浓度的OA诱导建立细胞脂肪化模型较为适宜,Gen与PPAR激动剂都能调节细胞的脂质代谢活性,调控细胞的TG含量。Gen可以通过代谢性核受体途径调节ATGL、HSL的活性,TNF-α、Leptin能够明显降低ATGL mRNA的表达,Gen是通过调控PPARs途径来抑制TNF-α、Leptin分泌,调控体内脂质代谢产生降脂效果,具有重要的生理意义。
[Abstract]:The liver is the main organ and important place of lipid metabolism. Fatty liver is one of the common liver diseases. Soybean isoflavones have been detected to have a significant therapeutic effect on experimental fatty liver in rats. Soybean isoflavone is a natural secondary metabolite isolated from the roots of legumes. Genistein Genin played an important role in the activity of soybean isoflavones. It is found that genistein plays an important physiological role in lipid metabolism mainly through the regulation of PPARs peroxisome proliferator activated receptor and the regulation of metabolic enzyme expression and activity. Objective: to explore the pathway of trihydroxyisoflavones regulating lipid metabolism through cell culture in vitro. To study the regulation and expression of Gen on the expression of peroxisome proliferator activated receptor (PPAR), liver X receptor (LXRN), adipose Triglyceride lipase (ATGL) and hormone sensitive triglyceride lipase (Hormone-sensitive lipase). To compare the effects of genistein and PPAR agonist on cell activity, and to explore the regulation of Gen on human tumor necrosis factor TNF- 伪 and leptin Leptin in adipogenic cells, it is suggested that trihydroxyisoflavone is in nutritional care and clinical pharmacology. To provide theoretical basis and experimental basis for regulating fat and protecting liver. Methods: human normal hepatocytes L-02, human hepatoma HepG2 Hep3b and mouse preadipocytes 3T3-L1 were cultured in vitro. The cells were divided into blank group, model group and PPAR agonist group. At first, MTT was used to determine the cell activity at different concentrations of oleic acid, to determine the optimal concentration of OA induction, and to establish a model of cell adipogenesis. The cell apoptosis was detected by flow cytometry with Annexin V/PI double staining method, and the toxicity of Gen compared with PPAR agonist and inhibitor was further verified. Four kinds of fatty cells were treated with genistein PPAR activator and inhibitor, and the mRNA expression of ATGL HSLPAR- 伪 PPAR- 伪 PPAR- 纬 and liver X receptor LXR were measured by real-time fluorescence quantitative PCR. Enzyme linked immunosorbent assay (Elisa) was used to detect the changes of TNF- 伪 Leptin in four kinds of lipopolysaccharide cell cultures treated with genistein PPAR activator and inhibitor. Results when the concentration of oleic acid was higher than 1mM, the cell viability was significantly decreased, and some cells were floating and necrotic. The TG content in the cell accumulated significantly. When oleic acid concentration was lower than 1mM, the cell viability was similar. The results of flow cytometry showed that genistein had protective effect on cell adipogenesis and increased the activity of PPARs and mRNA expression of LXR 伪, and the regulation of PPARs was more significant than that of LXR 伪. At the same time, the expression of m RNA in HSL and ATGL was also increased, and the concentration of TNF- 伪 Leptin could be regulated in a dose-dependent manner. Conclusion: the model of cell adipogenesis induced by OA at the concentration of 1mM is more suitable for regulating the lipid metabolism activity of cells by both PPAR agonists and genistein. Regulation of TG content. Gen can regulate the activity of TNF- 伪 Leptin through metabolic nuclear receptor pathway. TNF- 伪 -Leptin can significantly reduce the expression of ATGL mRNA by regulating the PPARs pathway to inhibit the secretion of TNF- 伪 -leptin, and to regulate the lipid metabolism in vivo to produce lipid-lowering effect. It has important physiological significance.
【学位授予单位】：合肥工业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R575.5

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