胆汁酸核受体激动剂对脂肪因子及其受体在非酒精性脂肪性肝病中的影响
发布时间：2018-09-12 13:11
【摘要】：研究背景和目的 随着肥胖发病率的上升,非酒精性脂肪性肝病(nonalcoholic fatty liver disease,NAFLD)现已成为了世界范围内最常见的慢性肝脏疾病之一,其发病率是其他常见慢性肝病的几倍,美国等西方国家发病率尤为高。而近些年来随着我国生活水平的提高,我国发病率也处于一个逐年上升的阶段,接近西方国家。NAFLD是在无过量酒精摄入的情况下患者肝细胞内的脂肪沉积,是由一系列疾病组成的一个疾病谱,包括单纯性脂肪肝、非酒精性脂肪性肝炎、脂肪性肝硬化等,被认为是代谢综合征在肝脏的一种表现,具有一系列与代谢综合征相似的表现,如胰岛素抵抗、血脂异常、高血压等,但其发病机制现在还不十分清楚,目前认为胰岛素抵抗是其发病过程中的关键因素。目前,NAFLD尚无临床证明有效的药物,唯一有效的治疗方法就是及早发现疾病,通过减肥及生活方式的改变达到治疗的目的,因此提醒我们开发研制针对NAFLD有效药物的必要性及紧迫性。 胆汁酸核受体,又被称为类法尼酯X受体(Farnesoid X receptor, FXR),属于核受体超家族的一员,高表达于肝脏、胃肠道、肾脏和肾上腺等器官,而心脏、脂肪组织则呈低表达状态。最早人们发现FXR可调节胆汁酸的稳态,随着进一步的研究,人们发现FXR还具有多种新的功能,其中最为重要的可能是其对代谢紊乱疾病包括NAFLD、糖尿病、高血压等的调节作用。FXR可通过多种途径调节]NAFLD的发生、发展过程,例如FXR可改善外周组织的胰岛素抵抗及通过调节肝脏的糖异生和肝糖分解过程控制葡萄糖稳态,可通过多种途径减少血脂水平从而缓解肝脏甘油三酯积聚、氧化应激及脂质过氧化等,还可以对抗肝细胞的炎症过程及改善肝脏纤维化状态,而炎症和纤维化正是非酒精性脂肪性肝炎重要的病理改变。值得注意的是,NAFLD患者肝脏的FXR表达水平较正常人低,提示FXR的低表达状态可能在NAFLD的发生发展中起着某些作用。结合FXR的以上特点,提示激活FXR可能会成为治疗NAFLD的一种理想手段。 白色脂肪组织是成年人身体中最丰富的组织,也是最大的内分泌器官,可分泌多种脂肪因子,包括脂联素、瘦素、抵抗素等,而各种脂肪因子通过与全身各组织器官发生联系,共同维持人体内的代谢平衡。脂肪因子参与调节胰岛素抵抗、能量代谢及代谢综合征的发生过程,也在NAFLD的发病机制中发挥着重要的作用,如脂联素是一种具有胰岛素增敏作用的脂肪因子,具有抗炎、抗动脉粥样硬化、抗糖尿病等作用,可减缓NAFLD的进程;瘦素可能具有维持糖代谢稳态、保护肝细胞、减少脂肪肝的作用；而抵抗素则可引起糖代谢紊乱、胰岛素抵抗、脂肪肝等。一些研究显示NAFLD患者脂肪因子受体的表达也出现了异常,意味着这些受体也可能参与了NAFLD的发病过程。 Giovanni Rizzo等证实了FXR的激活可促进脂肪细胞的分化过程,提示激活FXR可能对脂肪因子及其受体有一定的调节作用,但目前为止,较少实验研究FXR的激活对脂肪因子及其受体的影响。因此本实验的目的即证实FXR的激活是否可通过直接或间接地调控脂肪因子及其受体的表达,从而达到减缓NAFLD发生、发展的目的。本实验通过人工合成的高选择性的FXR动剂GW4064来影响脂肪细胞分化成熟的整个过程,研究GW4064对脂肪细胞的脂肪因子(包括脂联素、瘦素、抵抗素)及其对应受体(脂联素受体1(adiponectin receptor1,AdipoRl)、脂联素受体2(adiponectin receptor2, AdipoR2)和长型瘦素受体(long form leptin receptor, OB-R))的影响,同时,还研究了GW4064对脂肪因子作用的靶器官肝脏的脂肪因子受体(AdipoR2和OB-Rb)的影响。因此,本实验拟从分泌脂肪因子的脂肪细胞及脂肪因子作用的靶细胞-肝脏细胞系统地了解FXR的激活是否能直接或间接调节脂肪因子及其受体表达。 方法 1、HepG2细胞和3T3-L1前脂肪细胞的培养。用含10%的胎牛血清、100U/ml的青霉素、100μg/ml的链霉素的DMEM培养基培养于37℃、5%CO2环境中。 2、HepG2细胞和3T3-L1前脂肪细胞分组。3T3-L1前脂肪细胞分组：GW4064组于分化第0天加入5μmol/L GW4064至分化第8天,对照组则加入等体积的DMSO(因GW4064是用DMSO溶解的),分别取分化第0、2、4、6、8天的细胞组织和上清液检测。HepG2细胞分组：GW4064组加入5μmol/LGW4064,对照组则加入等体积的DMSO,分别取刺激第0、12、24、48小时的细胞组织和上清液检测。 3、3T3-L1前脂肪细胞诱导分化为成熟的脂肪细胞。待细胞汇合至100%后,继续培养2天,此时细胞退出生长周期,开始加入分化诱导液Ⅰ(含有0.5mmol/L1-甲基-3-异丁基黄嘌呤(1-methyl-3-isobutyl-xanthin,IBMX),1μmol/L地塞米松,5μg/mL胰岛素的完全培养基,此即为分化第0天,记为DO)。分化诱导液Ⅰ处理48小时(即分化第2天,记为D2)后,换成分化诱导液Ⅱ(含5μg/ml胰岛素的完全培养液),分化诱导液Ⅱ处理48小时(即分化第4天,记为D4)后,换成正常的完全培养基,每2天换一次液,待分化至第8天(记为D8),进行油红O染色,拍照。 4、实时荧光定量PCR反应(Quantitative Real time PCR,QRT-PCR)检测3T3-L1前脂肪细胞分化过程中PPAR-γ2.脂肪因子(脂联素、瘦素、抵抗素)及其受体(AdipoR1、AdipoR2、OB-Rb)mRNA的表达情况及GW4064对上述基因的影响；检测GW4064对HepG2细胞中脂肪因子受体(AdipoR2、OB-Rb) mRNA表达的影响。按照说明书,进行总RNA的提取、RNA定量和纯度检测、逆转录合成cDNA和PCR反应。扩增后得到相关数据和曲线,按相对定量2-△△Ct法分析得出结果。 5、酶联免疫吸附法(Enzyme linked immunosorbent assay,ELISA)检测3T3-L1前脂肪细胞分化过程中上清液中脂肪因子(脂联素、抵抗素和瘦素)的含量及GW4064对其影响：严格按照ELISA试剂盒说明书步骤操作,用酶标仪在450nm波长下测定吸光度(OD)值,得出标准曲线,计算样品浓度。 6、免疫蛋白质印迹(Western blot)检测3T3-L1前脂肪细胞分化过程中PPAR-γ2和脂肪因子受体(AdipoRl、AdipoR2和OB-Rb)的蛋白表达情况及GW4064对其表达的影响；检测GW4064对HepG2细胞中脂肪因子受体(AdipoR2和OB-Rb)表达的影响。步骤包括细胞总蛋白的提取、测定蛋白浓度、电泳、转膜、免疫反应、曝光显影。 7、数据统计。数据统计分析采用SPSS13.0软件,所有计量资料均表示为均数±标准差(x±s)。两组样本间比较采用两样本的t检验；多组样本间比较需先进行方差齐性检验,方差齐,采用单因素方差分析(one-way ANOVA);方差不齐,则采用校正的F检验(Welch法)。P0.05被认为差异具有统计学意义。所有实验均重复3次。 结果 1.3T3-L1前脂肪细胞的分化成熟。经过分化诱导液Ⅰ、Ⅱ的作用,3T3-L1前脂肪细胞在分化第2天即可见到细胞由梭形向圆形变化,分化第4天可见细胞变圆的同时细胞内有脂滴形成,分化第8天可见到75%-85%细胞变圆、内含大量脂滴、油红O染为红色,即为分化成熟的脂肪细胞。 2. QRT-PCR检测3T3-L1前脂肪细胞分化过程中PPAR-γ2、脂肪因子(脂联素、瘦素、抵抗素)及其受体(AdipoR1、AdipoR2、OB-Rb)mRNA的表达情况及GW4064对上述基因的影响。在3T3-L1前脂肪细胞未分化时,瘦素、抵抗素、PPAR-γ2及脂肪因子受体(AdipoR1、AdipoR2和OB-Rb)mRNA即可被检测到,而脂联素在分化第4天才可被检测到,并且上述基因均随着脂肪细胞的分化成熟表达量逐渐增加,其中脂联素、瘦素、AdipoR2、PPAR-γ2在经过GW4064刺激后表达量较对照组明显升高,差异具有统计学意义(P0.05)。 3. QRT-PCR检测GW4064对HepG2细胞中脂肪因子受体(AdipoR2和OB-Rb)mRNA的表达的影响。GW4064刺激后AdipoR2、OB-Rb mRNA相对表达量呈时间依赖性的增加,差异具有统计学意义(P0.05)。 4.ELISA检测脂肪因子(脂联素、瘦素和抵抗素)在3T3-L1前脂肪细胞分化过程中蛋白的表达情况及GW4064对其表达的影响。脂联素、瘦素、抵抗素均随着细胞的分化成熟蛋白表达量逐渐增加,且经过GW4064刺激后,脂联素、瘦素表达量显著高于对照组(P0.05),而抵抗素则无明显改变。 5.Western blot检测3T3-L1前脂肪细胞分化过程中PPARγ2及脂肪因子受体(AdipoR1、AdipoR2和OB-Rb)的蛋白表达情况及GW4064对其表达的影响。PPARγ2、AdipoR1、AdipoR2和OB-Rb均随着3T3-L1前脂肪细胞的分化成熟表达量逐渐增加,其中,GW4064刺激后PPARγ2的蛋白表达显著高于对照组(P0.05),而AdipoR1、AdipoR2和OB-Rb则无明显改变。 6.Western blot检测GW4064对HepG2细胞中脂肪因子受体(AdipoR2和OB-Rb)蛋白表达情况的影响。在HepG2细胞中,AdipoR2在经过GW4064刺激后蛋白表达量呈时间依赖性的增加(P0.05)；而OB-Rb则无明显改变。 结论 本研究证实FXR激动剂GW4064刺激3T3-L1前脂肪细胞可以上调其PPAR-γ2、脂联素、瘦素及AdipoR2mRNA的表达,同时可以促进PPAR-γ2、脂联素、瘦素蛋白的表达；另外,GW4064刺激HepG2细胞可上调其AdipoR2及OB-Rb mRNA的表达,并促进AdipoR2蛋白的表达。上述结果表明,FXR激动剂可影响脂肪细胞中某些脂肪因子及其受体的表达,亦可影响肝细胞的某些脂肪因子受体的表达。我们的实验结果还提示FXR激动剂对脂肪因子及其相关受体的影响可能通过或者部分通过诱导PPAR-γ的表达而实现的,后者可调节脂肪细胞分化过程中多种基因的表达。因脂肪因子及其受体在NAFLD的发生、发展中发挥着关键作用,提示FXR激动剂对NAFLD的作用可能是通过影响脂肪因子及其受体而实现的,这为进一步了解FXR对NAFLD的作用机制及其成为NAFLD的有效药物的可能性提供了理论基础。
[Abstract]:Research background and purpose
With the increasing incidence of obesity, non-alcoholic fatty liver disease (NAFLD) has become one of the most common chronic liver diseases in the world. The incidence of NAFLD is several times that of other common chronic liver diseases, especially in the United States and other Western countries. NAFLD is a fatty deposit in the hepatocytes of patients without excessive alcohol intake. It is a spectrum of diseases, including simple fatty liver disease, non-alcoholic fatty liver disease, fatty liver cirrhosis and so on. It is considered to be metabolic synthesis. One of the manifestations of hepatic syndrome has a series of similar manifestations with metabolic syndrome, such as insulin resistance, dyslipidemia, hypertension, etc. However, the pathogenesis is still unclear. Insulin resistance is considered to be the key factor in the pathogenesis of hepatic syndrome. The method is to discover the disease as early as possible and achieve the goal of treatment through weight loss and lifestyle changes. Therefore, it reminds us of the necessity and urgency of developing effective drugs for NAFLD.
Bile acid receptors, also known as Farnesoid X receptors (FXR), are a member of the nuclear receptor superfamily. They are highly expressed in the liver, gastrointestinal tract, kidneys and adrenal glands, while low-expression in the heart and adipose tissue. FXR also has a variety of new functions, the most important of which may be its regulatory role in metabolic disorders including NAFLD, diabetes, hypertension and so on. Glucose homeostasis can alleviate triglyceride accumulation, oxidative stress and lipid peroxidation in the liver by reducing blood lipid levels in various ways. It can also resist inflammation of hepatocytes and improve liver fibrosis. Inflammation and fibrosis are important pathological changes in NAFLD. The expression of FXR in the liver of the patients was lower than that of the normal subjects, suggesting that the low expression of FXR may play a role in the development of NAFLD.
White adipose tissue is the most abundant tissue and the largest endocrine organ in adults. It secretes many kinds of adipocytokines, including adiponectin, leptin, resistin and so on. All kinds of adipocytokines maintain the metabolic balance of the human body by contacting with various tissues and organs of the whole body. Adiponectin is an insulin-sensitized adipokine with anti-inflammatory, anti-atherosclerosis, anti-diabetes and other effects, which can slow down the process of NAFLD; leptin may maintain glucose metabolism homeostasis and protect liver cells. Some studies have shown abnormal expression of adipokine receptors in NAFLD patients, suggesting that these receptors may also be involved in the pathogenesis of NAFLD.
Giovanni Rizzo et al confirmed that activation of FXR can promote the differentiation of adipocytes, suggesting that activation of FXR may regulate adipocytokines and their receptors to some extent, but so far, few experiments have studied the effect of FXR activation on adipocytokines and their receptors. In this study, we investigated the effects of GW4064 on adipocyte adipokines (including adiponectin, leptin, resistin) and their corresponding receptors in adipocytes by affecting the whole process of adipocyte differentiation and maturation by synthesizing highly selective FXR agonist GW4064. The effects of GW4064 on adiponectin receptor 1 (AdipoRl), adiponectin receptor 2 (AdipoR2) and long form leptin receptor (OB-R) were also studied. The effects of GW4064 on adiponectin receptor 2 (AdipoR2) and OB-Rb (long form leptin receptor, OB-R) in the liver, the target organs of adipokines, were also studied. Adipocytes secreting adipokines and hepatocytes, the targets of adipokines, systematically understand whether FXR activation directly or indirectly regulates the expression of adipokines and their receptors.
Method
1, HepG2 cells and 3T3-L1 preadipocytes were cultured in DMEM medium containing 10% fetal bovine serum, 100U/ml penicillin and 100ug/ml streptomycin at 37 C and 5% CO2.
2. HepG2 cells and 3T3-L1 preadipocytes. 3T3-L1 preadipocyte group: GW4064 group was added 5 micromol/L GW4064 on the 0th day of differentiation to the 8th day of differentiation, while the control group was added DMSO of equal volume (because GW4064 was dissolved in DMSO). Cell tissues and supernatant of the 0th, 2nd, 4th, 6th and 8th day of differentiation were obtained and detected respectively. Cell tissues and supernatants were obtained at 0, 12, 24 and 48 hours of stimulation respectively.
3,3T3-L1 preadipocytes were induced to differentiate into mature adipocytes. After confluence to 100%, the cells were cultured for 2 days. At this time, the cells withdrew from the growth cycle and began to add differentiation inducing medium I (containing 0.5mmol/L 1-methyl-3-isobutyl-xanthin, IBMX), 1 micromol/L dexamethasone, 5 microgram/ml insulin) into the complete medium. Differentiation induction fluid I was treated for 48 hours (i.e. the second day of differentiation, denoted as D2), then changed into differentiation induction fluid II (complete medium containing 5 ug/ml insulin), treated for 48 hours (i.e. the fourth day of differentiation, denoted as D4), and changed into normal complete medium every 2 days until the eighth day (record) For D8), oil red O was stained and photographed.
4. Quantitative Real-time PCR (QRT-PCR) was used to detect the expression of PPAR-gamma 2. Adiponectin (adiponectin, leptin, resistin) and its receptor (AdipoR1, AdipoR2, OB-Rb) mRNA during the differentiation of 3T3-L1 preadipocytes, and the effect of GW4064 on the expression of Adipo receptor (Adipo receptor) in HepG2 cells. R2, OB-Rb) mRNA expression. According to the instructions, total RNA extraction, RNA quantitative and purity detection, reverse transcription synthesis of cDNA and PCR reaction. After amplification, the relevant data and curves were obtained, according to the relative quantitative 2-delta CT analysis results.
5. Enzyme linked immunosorbent assay (ELISA) was used to detect the adiponectin (adiponectin, resistin and leptin) content in the supernatant of 3T3-L1 preadipocyte during differentiation and the effect of GW4064 on it. The absorbance (OD) value was determined by enzyme-linked immunosorbent assay (ELISA) at 450 nm. The standard curve is used to calculate the sample concentration.
6. Western blot was used to detect the expression of PPAR-gamma 2 and adipokine receptors (AdipoRl, AdipoR2 and OB-Rb) during the differentiation of 3T3-L1 preadipocytes and the effect of GW4064 on the expression of adipokine receptors (AdipoR2 and OB-Rb) in HepG2 cells. Extraction, determination of protein concentration, electrophoresis, transfer membrane, immune reaction, exposure development.
7. Data statistics. SPSS13.0 software was used for statistical analysis of data. All measurement data were expressed as mean (+ standard deviation) (x (+ s). The comparison between two groups of samples was conducted by t test of two samples; the comparison between multiple groups of samples was conducted by homogeneity test of variance, homogeneity of variance, one-way ANOVA; and the variance was not homogeneous, F test was used for correction. The Welch (.P0.05) method was considered to be statistically significant. All experiments were repeated 3 times.
Result
1.3T3-L1 preadipocytes differentiated and matured. After the effect of differentiation inducer I and II, 3T3-L1 preadipocytes changed from spindle to round on the 2nd day of differentiation. On the 4th day of differentiation, fat droplets formed in the cells while the cells became round. On the 8th day of differentiation, 75% - 85% of the cells became round, containing a large number of lipid droplets and oil red O stained red. Color is the differentiation of mature adipocytes.
2. QRT-PCR was used to detect the expression of PPAR-gamma 2, adiponectin (adiponectin, leptin, resistin) and its receptor (AdipoR1, AdipoR2, OB-Rb) mRNA during the differentiation of 3T3-L1 preadipocytes and the effect of GW4064 on the above genes. NA could be detected immediately, and adiponectin could be detected only on the 4th day of differentiation, and the expression of the above genes increased gradually with the differentiation and maturation of adipocytes. The expression of adiponectin, leptin, AdipoR2 and PPAR-gamma-2 increased significantly after GW4064 stimulation compared with the control group (P 0.05).
3. The effect of GW4064 on the expression of adipokine receptor (AdipoR2 and OB-Rb) mRNA in HepG2 cells was detected by QRT-PCR. The relative expression of AdipoR2 and OB-Rb mRNA increased in a time-dependent manner after GW4064 stimulation, and the difference was statistically significant (P 0.05).
4. ELISA was used to detect the expression of adiponectin (adiponectin, leptin and resistin) during the differentiation of 3T3-L1 preadipocytes and the effect of GW4064 on the expression of adiponectin, leptin and resistin. Group (P0.05), but resistin did not change significantly.
5. Western blot was used to detect the expression of PPAR-gamma-2 and adipokine receptors (AdipoR1, AdipoR2 and OB-Rb) during the differentiation of 3T3-L1 preadipocytes and the effect of GW4064 on the expression of PPAR-gamma-2, AdipoR1, AdipoR2 and OB-Rb. The expression was significantly higher than that in the control group (P0.05), while AdipoR1, AdipoR2 and OB-Rb did not change significantly.
6. Western blot was used to detect the effect of GW4064 on the expression of adipokine receptors (AdipoR2 and OB-Rb) in HepG2 cells.
conclusion
This study confirmed that FXR agonist GW4064 stimulated 3T3-L1 preadipocytes to up-regulate the expression of PPAR-gamma-2, adiponectin, leptin and AdipoR2 mRNA, and to up-regulate the expression of PPAR-gamma-2, adiponectin and leptin. In addition, GW4064 stimulated HepG2 cells to up-regulate the expression of AdipoR2 and OB-Rb mRNA, and promoted the expression of AdipoR2 protein. Our results also suggest that FXR agonists may affect the expression of some adipocytokines and their receptors in adipocytes and some adipocytokine receptors in hepatocytes. Our results also suggest that the effects of FXR agonists on adipocytokines and their receptors may be mediated by or in part by inducing the expression of PPAR-gamma, the latter. Fatty factor and its receptor play a key role in the development of NAFLD, suggesting that the effect of FXR agonists on NAFLD may be achieved by affecting the expression of Adipocyte Factor and its receptor. This will help us to understand the mechanism of FXR on NAFLD and its role in NAFLD. The possibility of effective drugs provides a theoretical basis.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R575.5
本文编号：2239104
[Abstract]:Research background and purpose
With the increasing incidence of obesity, non-alcoholic fatty liver disease (NAFLD) has become one of the most common chronic liver diseases in the world. The incidence of NAFLD is several times that of other common chronic liver diseases, especially in the United States and other Western countries. NAFLD is a fatty deposit in the hepatocytes of patients without excessive alcohol intake. It is a spectrum of diseases, including simple fatty liver disease, non-alcoholic fatty liver disease, fatty liver cirrhosis and so on. It is considered to be metabolic synthesis. One of the manifestations of hepatic syndrome has a series of similar manifestations with metabolic syndrome, such as insulin resistance, dyslipidemia, hypertension, etc. However, the pathogenesis is still unclear. Insulin resistance is considered to be the key factor in the pathogenesis of hepatic syndrome. The method is to discover the disease as early as possible and achieve the goal of treatment through weight loss and lifestyle changes. Therefore, it reminds us of the necessity and urgency of developing effective drugs for NAFLD.
Bile acid receptors, also known as Farnesoid X receptors (FXR), are a member of the nuclear receptor superfamily. They are highly expressed in the liver, gastrointestinal tract, kidneys and adrenal glands, while low-expression in the heart and adipose tissue. FXR also has a variety of new functions, the most important of which may be its regulatory role in metabolic disorders including NAFLD, diabetes, hypertension and so on. Glucose homeostasis can alleviate triglyceride accumulation, oxidative stress and lipid peroxidation in the liver by reducing blood lipid levels in various ways. It can also resist inflammation of hepatocytes and improve liver fibrosis. Inflammation and fibrosis are important pathological changes in NAFLD. The expression of FXR in the liver of the patients was lower than that of the normal subjects, suggesting that the low expression of FXR may play a role in the development of NAFLD.
White adipose tissue is the most abundant tissue and the largest endocrine organ in adults. It secretes many kinds of adipocytokines, including adiponectin, leptin, resistin and so on. All kinds of adipocytokines maintain the metabolic balance of the human body by contacting with various tissues and organs of the whole body. Adiponectin is an insulin-sensitized adipokine with anti-inflammatory, anti-atherosclerosis, anti-diabetes and other effects, which can slow down the process of NAFLD; leptin may maintain glucose metabolism homeostasis and protect liver cells. Some studies have shown abnormal expression of adipokine receptors in NAFLD patients, suggesting that these receptors may also be involved in the pathogenesis of NAFLD.
Giovanni Rizzo et al confirmed that activation of FXR can promote the differentiation of adipocytes, suggesting that activation of FXR may regulate adipocytokines and their receptors to some extent, but so far, few experiments have studied the effect of FXR activation on adipocytokines and their receptors. In this study, we investigated the effects of GW4064 on adipocyte adipokines (including adiponectin, leptin, resistin) and their corresponding receptors in adipocytes by affecting the whole process of adipocyte differentiation and maturation by synthesizing highly selective FXR agonist GW4064. The effects of GW4064 on adiponectin receptor 1 (AdipoRl), adiponectin receptor 2 (AdipoR2) and long form leptin receptor (OB-R) were also studied. The effects of GW4064 on adiponectin receptor 2 (AdipoR2) and OB-Rb (long form leptin receptor, OB-R) in the liver, the target organs of adipokines, were also studied. Adipocytes secreting adipokines and hepatocytes, the targets of adipokines, systematically understand whether FXR activation directly or indirectly regulates the expression of adipokines and their receptors.
Method
1, HepG2 cells and 3T3-L1 preadipocytes were cultured in DMEM medium containing 10% fetal bovine serum, 100U/ml penicillin and 100ug/ml streptomycin at 37 C and 5% CO2.
2. HepG2 cells and 3T3-L1 preadipocytes. 3T3-L1 preadipocyte group: GW4064 group was added 5 micromol/L GW4064 on the 0th day of differentiation to the 8th day of differentiation, while the control group was added DMSO of equal volume (because GW4064 was dissolved in DMSO). Cell tissues and supernatant of the 0th, 2nd, 4th, 6th and 8th day of differentiation were obtained and detected respectively. Cell tissues and supernatants were obtained at 0, 12, 24 and 48 hours of stimulation respectively.
3,3T3-L1 preadipocytes were induced to differentiate into mature adipocytes. After confluence to 100%, the cells were cultured for 2 days. At this time, the cells withdrew from the growth cycle and began to add differentiation inducing medium I (containing 0.5mmol/L 1-methyl-3-isobutyl-xanthin, IBMX), 1 micromol/L dexamethasone, 5 microgram/ml insulin) into the complete medium. Differentiation induction fluid I was treated for 48 hours (i.e. the second day of differentiation, denoted as D2), then changed into differentiation induction fluid II (complete medium containing 5 ug/ml insulin), treated for 48 hours (i.e. the fourth day of differentiation, denoted as D4), and changed into normal complete medium every 2 days until the eighth day (record) For D8), oil red O was stained and photographed.
4. Quantitative Real-time PCR (QRT-PCR) was used to detect the expression of PPAR-gamma 2. Adiponectin (adiponectin, leptin, resistin) and its receptor (AdipoR1, AdipoR2, OB-Rb) mRNA during the differentiation of 3T3-L1 preadipocytes, and the effect of GW4064 on the expression of Adipo receptor (Adipo receptor) in HepG2 cells. R2, OB-Rb) mRNA expression. According to the instructions, total RNA extraction, RNA quantitative and purity detection, reverse transcription synthesis of cDNA and PCR reaction. After amplification, the relevant data and curves were obtained, according to the relative quantitative 2-delta CT analysis results.
5. Enzyme linked immunosorbent assay (ELISA) was used to detect the adiponectin (adiponectin, resistin and leptin) content in the supernatant of 3T3-L1 preadipocyte during differentiation and the effect of GW4064 on it. The absorbance (OD) value was determined by enzyme-linked immunosorbent assay (ELISA) at 450 nm. The standard curve is used to calculate the sample concentration.
6. Western blot was used to detect the expression of PPAR-gamma 2 and adipokine receptors (AdipoRl, AdipoR2 and OB-Rb) during the differentiation of 3T3-L1 preadipocytes and the effect of GW4064 on the expression of adipokine receptors (AdipoR2 and OB-Rb) in HepG2 cells. Extraction, determination of protein concentration, electrophoresis, transfer membrane, immune reaction, exposure development.
7. Data statistics. SPSS13.0 software was used for statistical analysis of data. All measurement data were expressed as mean (+ standard deviation) (x (+ s). The comparison between two groups of samples was conducted by t test of two samples; the comparison between multiple groups of samples was conducted by homogeneity test of variance, homogeneity of variance, one-way ANOVA; and the variance was not homogeneous, F test was used for correction. The Welch (.P0.05) method was considered to be statistically significant. All experiments were repeated 3 times.
Result
1.3T3-L1 preadipocytes differentiated and matured. After the effect of differentiation inducer I and II, 3T3-L1 preadipocytes changed from spindle to round on the 2nd day of differentiation. On the 4th day of differentiation, fat droplets formed in the cells while the cells became round. On the 8th day of differentiation, 75% - 85% of the cells became round, containing a large number of lipid droplets and oil red O stained red. Color is the differentiation of mature adipocytes.
2. QRT-PCR was used to detect the expression of PPAR-gamma 2, adiponectin (adiponectin, leptin, resistin) and its receptor (AdipoR1, AdipoR2, OB-Rb) mRNA during the differentiation of 3T3-L1 preadipocytes and the effect of GW4064 on the above genes. NA could be detected immediately, and adiponectin could be detected only on the 4th day of differentiation, and the expression of the above genes increased gradually with the differentiation and maturation of adipocytes. The expression of adiponectin, leptin, AdipoR2 and PPAR-gamma-2 increased significantly after GW4064 stimulation compared with the control group (P 0.05).
3. The effect of GW4064 on the expression of adipokine receptor (AdipoR2 and OB-Rb) mRNA in HepG2 cells was detected by QRT-PCR. The relative expression of AdipoR2 and OB-Rb mRNA increased in a time-dependent manner after GW4064 stimulation, and the difference was statistically significant (P 0.05).
4. ELISA was used to detect the expression of adiponectin (adiponectin, leptin and resistin) during the differentiation of 3T3-L1 preadipocytes and the effect of GW4064 on the expression of adiponectin, leptin and resistin. Group (P0.05), but resistin did not change significantly.
5. Western blot was used to detect the expression of PPAR-gamma-2 and adipokine receptors (AdipoR1, AdipoR2 and OB-Rb) during the differentiation of 3T3-L1 preadipocytes and the effect of GW4064 on the expression of PPAR-gamma-2, AdipoR1, AdipoR2 and OB-Rb. The expression was significantly higher than that in the control group (P0.05), while AdipoR1, AdipoR2 and OB-Rb did not change significantly.
6. Western blot was used to detect the effect of GW4064 on the expression of adipokine receptors (AdipoR2 and OB-Rb) in HepG2 cells.
conclusion
This study confirmed that FXR agonist GW4064 stimulated 3T3-L1 preadipocytes to up-regulate the expression of PPAR-gamma-2, adiponectin, leptin and AdipoR2 mRNA, and to up-regulate the expression of PPAR-gamma-2, adiponectin and leptin. In addition, GW4064 stimulated HepG2 cells to up-regulate the expression of AdipoR2 and OB-Rb mRNA, and promoted the expression of AdipoR2 protein. Our results also suggest that FXR agonists may affect the expression of some adipocytokines and their receptors in adipocytes and some adipocytokine receptors in hepatocytes. Our results also suggest that the effects of FXR agonists on adipocytokines and their receptors may be mediated by or in part by inducing the expression of PPAR-gamma, the latter. Fatty factor and its receptor play a key role in the development of NAFLD, suggesting that the effect of FXR agonists on NAFLD may be achieved by affecting the expression of Adipocyte Factor and its receptor. This will help us to understand the mechanism of FXR on NAFLD and its role in NAFLD. The possibility of effective drugs provides a theoretical basis.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R575.5
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2 王倩;管小琴;;非酒精性脂肪肝病与胰岛素抵抗[J];中国临床康复;2006年36期
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