【作者】 张颖玮；

【导师】 梅长林；

【作者基本信息】 第二军医大学 ， 内科学， 2008， 博士

【摘要】 肾脏衰老一直是临床与基础研究都非常感兴趣的问题,随着衰老,肾脏的形态以及功能都发生显著的改变,肾小球滤过率不依赖于明显的病理改变而逐渐下降,衰老导致了肾小球、血管、肾脏实质等一系列改变。诸如高血压、糖尿病都可以对老龄肾脏造成一定的损伤。我们的研究以肾脏衰老为突破口,以此发现器官衰老的主要环节,揭示器官衰老的一些共同机制,提出延缓衰老的有效措施。本课题拟在原有的研究基础上,从肾脏衰老入手,观察PPARγ在。肾脏表达的随龄变化情况,证实PPARγ在肾脏衰老中的作用。动物实验验证PPARγ激动剂罗格列酮治疗肾脏衰老的安全性和有效性,从氧化应激状态、信号传导调节细胞周期影响细胞分化等角度对其抗衰老的分子机制进行初步探讨,并运用代谢组学的技术开展延缓大鼠肾脏衰老的分析和生物标记物发现,将分子生物学与代谢组学技术结合起来,探索抗肾脏衰老的新方法、新途径。我们以3月、12月、24月自然衰老SD大鼠为模型,分别代表青年组、中年组、老年组。免疫组化显示PPARγ在各年龄组大鼠肾组织的肾小管、集合管上皮细胞中均有分布,主要分布于细胞核内,胞浆内也有少量表达,在老年大鼠皮质肾小球系膜区系膜细胞、偶尔在壁层上皮细胞内也有阳性染色。原位杂交结果进一步在mRNA水平上证实了这一结果。免疫印迹分析结果显示PPARγ蛋白在大鼠肾脏衰老过程中表达下降。SOD、GStt-Px两种衰老标志物在大鼠肾脏衰老过程中含量也出现下降,并且两组数据呈正相关。在第二部分研究里,以罗格列酮干预中年、老年大鼠12周,以能量限制为阳性对照组。发现PPARγ蛋白在中年、老年罗格列酮及限食组的大鼠肾脏中的表达均上调。作为衰老标志物的P16在中年、老年罗格列酮组及限食组均出现不同程度的降低。P53蛋白在老年及中年各组大鼠中表达差异均不明显。推测罗格列酮在增强动物抗氧化能力的同时,可能通过调节P16/Rb信号转导通路,下调P16的表达减轻对pRb的抑制,细胞周期阻滞缓解得以顺利进行,从而增强了中年、老年大鼠肾脏细胞的增生分化能力。另外在实验中还发现罗格列酮能降低中年及老年大鼠的血清胆固醇、甘油三酯水平,加强其分解对衰老肾脏起到保护作用。在第三部分实验中我们收集了老年对照组、罗格列酮组、能量限制组大鼠的血液样品,运用液质联用的代谢组学方法(HPLC/Ms),通过比较进入动物血清化学成分及代谢物谱,考察了罗格列酮延缓大鼠肾脏衰老的物质基础。发现各组图中主成分积分值基本集中分布于椭圆形散点图的几个区域,各组间无明显交叉和重叠。正常组与罗格列酮组、能量限制组之间有着代谢产物的不同,说明血浆代谢组学分析能够较好地反映罗格列酮干预组与正常对照组及能量限制组之间的差异。在图谱中可以发现肉毒碱(Carnitine)、植物鞘氨醇(Phytosphingosine)、棕榈酰磷脂胆碱(Palmitoyllysophosphatidylcholine)、甘油磷酰胆碱(stearoylglycerophosphocholine)等脂质化合物改变较为明显,这些发生改变的代谢物可以作为罗格列酮延缓老年大鼠肾脏衰老的生物标志物做进一步的研究。并且上述脂质化合物与能量代谢、脂肪代谢途径密切相关,这些代谢物的改变提示罗格列酮干预后动物能量代谢及脂肪代谢功能均发生了变化,这可能是老年大鼠肾脏衰老发生、发展的潜在原因,从而揭示了罗格列酮延缓肾脏衰老的生物学本质。综上所述我们认为PPARγ参与了大鼠肾脏衰老的调控,PPARγ激动剂罗格列酮可以增强老年动物抗氧化应激能力,通过抑制肿瘤抑制因子P16的表达而调节细胞周期、增强衰老肾脏细胞的增生分化能力,并能调节老年大鼠自身脂肪代谢紊乱,增加甘油三酯的分解,增强胰岛素敏感性,从而保护肾组织延缓了衰老。更多还原

【Abstract】 Aging of the kidney is a problem of clinical and basic interest.The glomerular filtration rate falls progressively,independent of overt pathology.Glomerular,vascular and accompanying parenchymal changes occur and other disorders associated with ageing,such as diabetes and hypertension,have a stochastic deleterious effect on both form and function.We focus our research on kidney aging to reveals the common mechanism and provide the effective measures.Present study will be based on the original research to observe the expression and the role of the PPARγduring the rat kidney aging.In the research,we attempt to document the safety and effectiveness of PPARγagonist rosiglitazone by animal experiment,and futher to explore the molecular regulation of PPARγduring the aging process.The beneficial effects of PPARγagonist antiaging can be analysized through the cellular signal transduction activities and the modulation of various redox-sensitive transcription factors.In addition,the effect of Rosiglitazone delaying kidney ageing in rat will be demonstrated by metabonomic technique and to dicover the biomarker of it.We will combine the molecular biology and metabonomics to provide a new method and pathway of preventing rat kidney aging.We made the 3month,12month and 24month natural aging rat as model which represent young,middle-aged and old group respectively.Significant expression of PPARγprotein were detected in the cell of renal tubule and collecting duct in each group by immunohistochemistry.PPARγprotein expression was observed as nuclear and cytoplasmic staining.In old age group PPARγprotein were also observed little in the mesangial cells and Bowman capsul’s epithelial cell.However,hybridization in situ also verified the above result at mRNA level.Westernblot result shows that PPARγprotein decreased with age,while the activity of superoxide dismutase(SOD) and Glutathione peroxidase(GSH-PX) in rat kidney were decreased.The activity of SOD and GSH-PX were positively correlated with the expressions of PPARγ.In the second part,we designated the calorie restriction group as positive control while treated the middle-aged rat group and old rat group with RGTZ.We find the expression of PPARγprotein increased in RGTZ and CR groups in middle-aged and old rat kidney tissue compared with control group.P53 protein were no obvious different among different groups. Collecting the serum samples of each old rat group,we confirmed the chemical componds and final products of metabolism in the rat serm by HPLC/MS analysis.The results declare that the principle component score was usually distributed in several regions of the scatterplot ellipse graph,and each group has no obvious acrossing.The metabolism products are significantly different among every group.In this study,5 biomarkers were structurally proposed and all these metabolites will provide more information to study the mechanism of preventing rat kidney senescence.In addition,the lipid componds are closely connected with energy and lipid metabolism.It is conceivable that all of the differences observed here investigate that the energy and lipid metabolism in animal have changed much,and perhaps it is the potential reason to induce aging.All above were summarized as following:PPARγparticipate in the regulating of rat kidney aging.Date further revealed that PPARγagonist enhance the old animal’s ability to anti-oxidative stress,reduce the expression of tumor inhibit factor P16. Preventing kidney aging of RGTZ,also provide a better understanding of the role of PPARγ,which can regulate the disorder of lipid metabolism,increase the breakdown of TG,and enhance the sensibility of insulin.更多还原