- PDF Free Download

Size: px

Start display at page:

Download ""

隐冀
7 years ago
Views:

1 学校编码 :10384 分类号密级学号 : UDC 硕士学位论文揭示离子霉素作为 PPARγ 配体对改善 2 型糖尿病的功能研究 Revealing Ionomycin as PPARγ ligands for improving 2 type diabetes 潘志福指导教师姓名 : 李勇教授专业名称 : 细胞生物学论文提交日期 : 2014 年 04 月论文答辩时间 : 2014 年 05 月学位授予日期 : 2014 年月答辩委员会主席 : 评阅人 : 2014 年 05 月

3 厦门大学学位论文原创性声明本人呈交的学位论文是本人在导师指导下, 独立完成的研究成果本人在论文写作中参考其他个人或集体已经发表的研究成果, 均在文中以适当方式明确标明, 并符合法律规范和厦门大学研究生学术活动规范 ( 试行 ) 另外, 该学位论文为 ( 李勇 ) 课题 ( 组 ) 的研究成果, 获得 ( 李勇 ) 课题 ( 组 ) 经费或实验室的资助, 在 ( 李勇 ) 实验室完成 ( 请在以上括号内填写课题或课题组负责人或实验室名称, 未有此项声明内容的, 可以不作特别声明 ) 声明人 ( 签名 ): 年月日

5 厦门大学学位论文著作权使用声明本人同意厦门大学根据中华人民共和国学位条例暂行实施办法等规定保留和使用此学位论文, 并向主管部门或其指定机构送交学位论文 ( 包括纸质版和电子版 ), 允许学位论文进入厦门大学图书馆及其数据库被查阅借阅本人同意厦门大学将学位论文加入全国博士硕士学位论文共建单位数据库进行检索, 将学位论文的标题和摘要汇编出版, 采用影印缩印或者其它方式合理复制学位论文本学位论文属于 : ( )1. 经厦门大学保密委员会审查核定的保密学位论文, 于年月日解密, 解密后适用上述授权 ( )2. 不保密, 适用上述授权 ( 请在以上相应括号内打或填上相应内容保密学位论文应是已经厦门大学保密委员会审定过的学位论文, 未经厦门大学保密委员会审定的学位论文均为公开学位论文此声明栏不填写的, 默认为公开学位论文, 均适用上述授权 ) 声明人 ( 签名 ): 年月日 3

7 目录目录 1 前言核受体简介核受体的分类与命名核受体结构 PPAR 受体研究进展 PPARs 受体的分布和功能 PPARγ 的特点 PPARγ 发挥作用的方式 PPARγ 的辅因子调节 PPARγ 的翻译后修饰 PPARγ 的功能 PPARγ 与脂肪细胞分化 PPARγ 与肿瘤 PPARγ 和炎症反应 PPARγ 和糖尿病 PPARγ 和脂类代谢 PPARγ 配体 PPARγ 与配体的结合方式 PPARγ 的内源性配体 PPARγ 的外源性配体及 TZDs 副作用 Ionomycin 简介 Ionomycin 分子结构验证 Ionomycin 是 PPARγ 特异性配体糖尿病模型小鼠简介实验方法实验使用的主要仪器小鼠生理实验小鼠血清成分的测定肝脏甘油三酯胆固醇的测定检测组织基因的表达提取肝脏和附睾脂肪垫的 mrna mrna 的反转录 Real time-pcr 检测基因表达 Real time-pcr 数据处理测定葡萄糖耐受性和胰岛素敏感性 ELISA 测定胰岛素 CDK 激酶活性检测 T3-L1 细胞分化 I

8 目录 2.5 MTT 法检测 Ionomycin 对细胞存活率的影响数据统计学分析实验结果 Ionomycin 对 db/db 小鼠体重和摄食量的影响 Ionomycin 对 db/db 小鼠血糖胰岛素及甘油三酯的影响 Ionomycin 对 db/db 小鼠肝脏甘油三酯和胆固醇的影响 Ionomycin 提高 db/db 小鼠葡萄糖耐受性和胰岛素敏感性 Ionomycin 对 db/db 小鼠脂类合成和糖异生基因表达的影响 Ionomycin 对 db/db 小鼠炎症基因表达的影响 Ionomycin 抑制 CDK5 对 PPARγ LBD Ser273 的磷酸化 Ionomycin 诱导 3T3-L1 细胞分化较弱 Ionomycin 基本不影响细胞存活率 Ionomycin 对水肿相关基因的表达的影响讨论参考文献致谢 II

9 Contents Contents Abstract (In Chinese) 1 Abstract (In English) 1 1 Introduction Introduction of nuclear receptor The name and classification of nuclear receptor The structure of nuclear receptor The research progress of PPAR The distribution and function of PPAR The characteristics of PPARγ The role of PPARγ PPARγ is regulated by cofactors The modification after the translation of PPARγ The function of PPARγ PPARγ and adipocyte differentiation PPARγ and tumor PPAR γ and inflammation PPAR γ and diabetes PPARγ and lipid metabolism PPAR ligands The combination way of the ligand to PPARγ The endogenous ligand of PPARγ The exogenous ligands of PPARγ and the side effects of TZDs Introduction of Ionomycin The molecular structure of Ionomycin Validating Ionomycin is a PPARγ specificity ligand Introduction of the diabetic model mice 21 III

10 Contents 2 Experimental methods The main instruments Physiological experiments in mouse Mouse serum components were measured Liver triglyceride, cholesterol were measured Gene expression analysis mrna extraction form liver tissues and epididymal fat pad Reverse transcription of mrna Real time PCR to determine gene expression Processing data Glucose tolerance and insulin sensitivity Serum insulin levels were determined by ELISA CDK kinase assay T3-L1 adipocyte differentiation The survival of cell was determined by MTT Data statistical analysis 34 3 Experimental results The effect of Ionomycin on diet and body weight of db/db mice Ionomycin had an influence on the levels of blood glucose, insulin, and triglycerides Ionomycin had an influence on the levels of liver s triglyceride and cholesterol Ionomycin improved glucose tolerance and insulin sensitivity The mrna levels of genes associated with lipid synthesis and gluconeogenesis were effected in epididymal fat pads The mrna levels of genes associated with inflammatory were effected Ionomycin blocked the phosphorylation of PPAR γ at Ser273 mediated by CDK Ionomycin did not induce 3T3-L1 pre-adipocyte differentiation 44 IV

11 Contents 3.9 Ionomycin had no effect on the survival of cells The mrna levels of genes associated with fluid retention such as ion channel and water channel were lowered in mimcd Discussion 47 5 References 56 6 Acknowlegements 67 V

13 摘要摘要核受体 PPARγ(peroxisome proliferator-activated receptor gamma: 过氧化酶增殖因子活化受体 γ) 在脂肪生成和葡萄糖代谢过程中发挥着关键的作用, 是 TZDs (thiazolidinedione) 类药物治疗糖尿病的分子靶点,TZDs 活化 PPARγ 后能改善胰岛素的敏感性, 降低血糖, 但会引发一些不良的副作用, 如体重增加水肿心血管疾病等我们通过 Alphascreen 手段筛选出 Ionomycin( 离子霉素, Streptomyces conglobatus 代谢产生的一种抗生素 ) 是 PPARγ 的配体,Ionomycin 能特异性结合 PPARγ LBD 并激活 PPARγ, 募集辅激活因子, 且抑制 PPARγ 与辅抑制因子结合, 诱导 PPARγ 转录活性, 前期蛋白晶体结构显示, 与其它配体相比,Ionomycin 与 PPARγ 的 LBD 有独特的结合方式本论文用 Ionomycin 药物处理 2 型糖尿病模型小鼠 :db/db, 结果发现 Ionomycin 显著降低小鼠血清中葡萄糖和胰岛素的水平, 提高小鼠的葡萄糖耐受性和胰岛素敏感性, 同时降低 db/db 小鼠肝脏胆固醇和甘油三酯含量, 对 db/db 小鼠附睾脂肪组织和肝脏组织的基因表达进行分析, 发现 Ionomycin 能抑制脂肪酸合成相关基因的表达, 因此不会诱导脂肪细胞合成脂肪酸, 并提高脂联素 ( 能增强胰岛素敏感性 ) 基因表达量, 同时能抑制肝脏中糖异生基因和炎症基因表达, 从而抑制糖异生和肝糖原的降解, 降低血糖另外,CDK5 与 P25 蛋白结合后被其活化进而磷酸化 PPARγ-S273, 能降低胰岛素敏感性, 而 Ionomycin 能在体内和体外抑制活化的 CDK5 磷酸化 PPARγ-S273, 表明 PPARγ 配体 Ionomycin 改善糖尿病与 CDK5 激酶信号通路有关通过 MTT 实验表明 Ionomycin 基本对 HepG2 Cos7 和 mimcd3 等细胞的存活率无影响, 同时降低 mimcd3 细胞离子通道基因和水通道基因等水肿基因的表达, 另外 Ionomycin 诱导脂肪细胞分化的能力与对照组相似而明显弱于 Rosiglitazone, 表明 Ionomycin 诱导脂肪分化能力很弱和不会促进小鼠肾内髓质收集管细胞对水的重吸收, 这与 Ionomycin 处理小鼠后无体重增加和脂肪垫比重增加的结果一致因此 Ionomycin 作为 PPARγ 的配体, 结合到 PPARγ LBD, 募集辅激活因子, 改善糖尿病的症状, 降低血糖和提高胰岛素敏感性且没有发现明显的副作用, 可为治疗糖尿病提供一种有效的药物模板或药物小分子关键词 : PPARγ; 离子霉素 ; 糖尿病 VI

15 Abstract Abstract The nuclear hormone receptor peroxisome proliferator-activated receptor γ (PPARγ) plays central roles in adipogenesis and glucose homeostasis and is the molecular target for the thiazolidinediones(tzds) class of antidiabetic drugs. Activation of PPARγ by TZDs improve insulin sensitivity, however, this is accompanied with the induction of several undesirable side effects, such as weight gain, fluid retention and cardiovascular disease, it is important to find PPAR γ ligands that retain the benefits in improving insulin resistance but with reduced side effects. We had identified that Ionomycin, an antibiotic produced by Streptomyces conglobatus metabolism, was a novel PPARγ ligand by Alphascreen assay. Ionomycin was able to specifically bind and activate PPARγ by recruiting coactivator and inhibiting the binding of auxiliary inhibitor, eventually induced PPARγ transcriptional activity. The crystal structure showed that Ionomycin interacted with the PPARγ ligand-binding domain in a unique binding mode with properties and epitopes distinct from those of TZD drugs. We explored the biological effects of Ionomycin on diabetic model mouse. Mice were treated with Ionomycin by intraperitoneal injection, the results showed that Ionomycin could significantly reduce blood glucose and insulin, it also reduced liver cholesterol and triglycerides, and improved glucose tolerance and insulin sensitivity. We analyzed the gene expression in epididymal adipose tissue and liver tissue from mice, Ionomycin suppressed the expression of genes associated with fatty acid synthesis and gluconeogenesis, but increased adiponectin (could enhance insulin sensitivity) gene expression. As a PPARγ ligand, Ionomycin did not induce adipocyte fatty acid synthesis and gluconeogenesis and glycogenolysis.furthermore, PPARγ Ser273 was phosphorylated by the activation of CDK5 (cyclin-dependent kinase 5) and led to reduce insulin sensitivity. Ionomycin effectively blocked the phosphorylation of PPAR γ at Ser273 by CDK5 both in vitro and in vivo, showed that the improving diabetes of Ionomycin was connected with CDK5 kinase signaling pathway. In addition, Ionomycin had no effects on the survival of HepG2, Cos7 and VII

16 Abstract mimcd3 cells by MTT assay, and it could reduce the expression of genes associated with fluid retention genes such as ion channel genes and water channel genes in mimcd3 cell. Meanwhile, the ability of Ionomycin to induce 3T3-L1 cell differentiation was obvious weaker than Rosiglitazone, suggested that Ionomycin neither induced adipogenesis and reabsorption of water capacity in mouse kidney medullary collecting duct cells, nor aroused other obvious side effects. As a result, Ionomycin bound to PPARγ LBD, recruited cofactor of PPARγ to improve the symptoms of diabetes and did not show obvious side effects, as a result, Ionomycin may represent a unique template for designing novel PPAR γ ligands with advantages over current TZD drugs. Key Words: PPARγ; Ionomycin; diabetes. VIII

17 Degree papers are in the Xiamen University Electronic Theses and Dissertations Database. Full texts are available in the following ways: 1. If your library is a CALIS member libraries, please log on and submit requests online, or consult the interlibrary loan department in your library. 2. For users of non-calis member libraries, please mail to etd@xmu.edu.cn for delivery details.

2

10384 -------- 9601008 UDC---------------------- 1999 1999 1999 ------------------------ ------------------------ 1 2 .4...4 7 14.14..18..22..22..24 3 4.27.32.39 1 2 3 4 5 6 7 8 9 10 11 5 12 6 1 2 3 4