二甲双胍在治疗草酸钙肾结石形成中的作用及机制研究

发布时间：2018-07-10 19:07

本文选题：肾结石 + 二甲双胍　；参考：《天津医科大学》2017年博士论文

【摘要】：目的:在世界范围内,肾结石的发病率正在逐年升高,而造成这一趋势的原因尚不清楚,因而近年来关于泌尿系结石发病原因和药物预防治疗的研究越来越受到广大临床医师和科研工作者的关注。天津医科大学第二医院尿石症治疗中心是我国华北地区最大的尿石症防治基地,为了系统评估我国华北地区泌尿系结石的不同结石成分的构成比例和分布特点,也为今后深入开展针对泌尿系结石发病机制的研究和药物治疗奠定基础,我们对我中心的尿石症临床资料进行了回顾性分析。在此基础上,我们针对我国最为常见结石成分,即草酸钙结石的预防和药物治疗开展了进一步的研究,我们选择价格低廉,易于推广普及,又具有多种临床应用潜能的二甲双胍药物作为我们的研究对象,系统评估其是否具有抑制草酸钙结石形成的作用,以及其发挥这一作用的具体机制。方法:1、本研究通过红外光谱法检测了2002年1月1日至2014年12月31日期间,天津医科大学第二医院尿石症治疗中心收集的2813例泌尿系结石样本,测定其具体的结石成分,并采用多因素Logistic回归分析对患者的结石成分分析结果和相关的临床资料的关联,以公历年、公历月、性别和年龄组(其中年龄按照0-29岁,30-39岁,40-49岁,50-59岁,60-69岁,70-79岁和80岁及以上进行分组)作为协变量,以每种类型的尿路结石的结石成分作为因变量。2、本研究探讨了二甲双胍对草酸诱导的肾小管上皮细胞损伤的预防作用和高草酸尿大鼠结石模型的肾结石形成的影响。本研究通过MTT检测明确二甲双胍和草酸钠对肾小管上皮细胞的细胞活性的影响,并进一步在体外细胞模型中检测了细胞内超氧化物歧化酶(SOD)活性和丙二醛(MDA)水平;此外,本研究通过乙二醇(EG)诱导雄性Sprague-Dawley大鼠构建草酸钙肾结石动物模型,实验分组为:对照组,EG处理组和EG+二甲双胍治疗组;在造模8周后,比较各组大鼠肾组织中氧化应激相关指标SOD和MDA水平的变化,以及草酸钙晶体的形成。3、本研究进一步探讨了二甲双胍在抑制草酸钙肾结石形成中对炎症调节因子的调控作用。本研究通过CCK-8检测了二甲双胍对MDCK和HK-2细胞的细胞毒性;随后,在体外实验中,本研究通过定量RT-PCR,western blot和ELISA检测检测了细胞内和细胞外的MCP-1和OPN的mRNA转录和蛋白表达情况;本研究通过乙二醇(EG)诱导雄性Sprague-Dawley大鼠构建草酸钙肾结石动物模型,实验分组为:对照组,EG处理组和EG+二甲双胍治疗组。在造模8周后,通过定量RT-PCR和western blot比较了各组大鼠肾组织中MCP-1和OPN的mRNA转录和蛋白表达及其与草酸钙肾结石形成的关系。结果:1、含钙结石最为常见,占总体的84.1%。女性患者更容易患CaP和MAP结石,而男性患者更容易患COM,COD和UA结石。老年患者UA结石的发病风险增加,而COD,CaP和胱氨酸结石的发病风险降低。此外,从2002年到2014年,COD和MAP结石呈增高趋势,而CaP,UA和胱氨酸结石呈下降趋势。公历月(季节)对结石成分的影响不显著。2、二甲双胍显著改善了草酸诱导的MDCK细胞和HK-2细胞的细胞毒性。此外,二甲双胍还显著缓解了草酸诱导的两种细胞的MDA水平的增加和SOD活性的降低。在体内研究中,与对照组相比,EG处理组MDA水平升高,SOD活性降低,而EG+二甲双胍治疗组的这些指标显著改善。与EG处理组相比,EG+二甲双胍治疗组肾结石形成明显减少。3、在体外,二甲双胍显著抑制草酸诱导的MCP-1和OPN的mRNA和蛋白质表达。在体内研究中,EG处理组与对照组相比,MCP-1和OPN的mRNA和蛋白表达显著增加,二甲双胍治疗能够显著改善它们的过表达,这一改善作用与草酸钙结石的形成呈一致的趋势。结论:1、本研究发现从2002年到2014年,我们发现COM是最为常见的结石成分,COD结石和MAP结石有增加的趋势,而CaP结石,UA结石和胱氨酸结石则有减少的趋势,泌尿系结石成分在不同性别和不同年龄组之间存在着显著的差异性。2、本研究二甲双胍可以有效缓解由草酸诱导的脂质过氧化产生及由此所导致的肾小管上皮细胞损伤。更为重要的是,本研究首次证实了二甲双胍具有治疗草酸钙肾结石的作用,并在体外和体内动物模型上确切地证实了二甲双胍能够通过抗氧化机制抑制肾结晶沉积。3、本研究还证实二甲双胍在治疗草酸钙肾结石的过程中能够在体内和体外均有效缓解草酸诱导肾小管上皮细胞炎症调节因子OPN和MCP-1的过表达。这些发现,使我们有理由相信二甲双胍有可能成为一种治疗和预防草酸钙肾结石的有效药物,并且这可能会大大造福于广大原发性高草酸尿症患者和复发性草酸钙肾结石患者。
[Abstract]:Objective: in the world, the incidence of renal calculi is increasing year by year, and the cause of this trend is still unclear. Therefore, the research on the causes of urinary calculi and the research of drug prevention and treatment have attracted more and more attention of clinicians and researchers in recent years. It is the largest base for the prevention and treatment of urolithiasis in North China. In order to systematically evaluate the proportion and distribution of different stones in the urinary calculi in North China, it also lays the foundation for the research and drug treatment for the pathogenesis of urolithiasis in the future. On the basis of this, we have further studied the prevention and drug treatment of calcium oxalate stone, which is the most common stone ingredient in our country. We choose the metformin, which is cheap, easy to popularize, and has a variety of clinical potential as our research object. The effect of inhibiting calcium oxalate stone formation and the specific mechanism of its exertion were found. Methods: 1. In this study, 2813 cases of urinary calculi collected from January 1, 2002 to December 31, 2014 in Second Hospital Affiliated to Tianjin Medical University were detected by infrared spectroscopy. Multiple factor Logistic regression analysis was used to analyze the correlation between the results of the stone composition analysis and the related clinical data in the group of the calendar year, the calendar month, the sex and the age group (aged 0-29, 30-39, 40-49, 50-59, 60-69, 70-79 and 80 years old) as a covariate, with each type of urinary calculi stone As a dependent variable.2, the effect of metformin on the prevention of renal tubular epithelial cell injury induced by oxalic acid and the formation of kidney stones in the calculus model of high oxalate rats were investigated in this study. The effect of metformin and sodium oxalate on the cell activity of renal tubular epithelial cells was determined by MTT and further in vitro In cell model, intracellular superoxide dismutase (SOD) activity and malondialdehyde (MDA) level were detected. In addition, this study induced the construction of calcium oxalate kidney stone animal model in male Sprague-Dawley rats by ethylene glycol (EG). The experimental group was divided into control group, EG treatment group and EG+ metformin treatment group. After 8 weeks of modeling, the kidney group was compared. The changes in the levels of oxidative stress related indicators SOD and MDA and the formation of.3 in calcium oxalate crystals. This study further explored the regulation of metformin on the inhibition of inflammatory regulatory factors in the formation of calcium oxalate nephrolithiasis. The cytotoxicity of metformin to MDCK and HK-2 cells was detected by CCK-8, and then in vitro experiments. In this study, quantitative RT-PCR, Western blot and ELISA were used to detect the mRNA transcriptional and protein expression of MCP-1 and OPN in cells and cells. This study induced the construction of calcium oxalate kidney stones in male Sprague-Dawley rats by ethylene glycol (EG). The experimental group was divided into the control group, the EG treatment group and the EG+ metformin treatment group. After 8 weeks of modeling, the mRNA transcriptional and protein expression of MCP-1 and OPN in kidney tissues of rats in each group were compared by quantitative RT-PCR and Western blot. Results: 1, calcium containing calcium stones were most common, and the overall 84.1%. female patients were more likely to suffer from CaP and MAP stones, while male patients were more likely to suffer from COM, COD, and UA nodes. The risk of UA stones in elderly patients increased, while the risk of COD, CaP and cystine stones decreased. In addition, from 2002 to 2014, COD and MAP stones increased, while CaP, UA and cystine stones declined. The influence of the calendar month (season) on the stone composition was not significant.2, and metformin significantly improved the oxalic acid induced MDCK. The cytotoxicity of cells and HK-2 cells. In addition, metformin also significantly alleviated the increase in MDA level of oxalic acid induced two cells and the decrease of SOD activity. In the study in vivo, the MDA level in the EG treatment group increased and the SOD activity decreased, while the EG+ metformin treatment group improved significantly. Compared with the EG treatment group, The formation of renal calculi in the EG+ metformin treatment group was significantly reduced by.3. In vitro, metformin significantly inhibited the mRNA and protein expression of MCP-1 and OPN induced by oxalic acid. In the study in vivo, the mRNA and protein expression of MCP-1 and OPN increased significantly compared with the control group, and the two metformin treatment could significantly improve their overexpression. The good effect is consistent with the formation of calcium oxalate stones. Conclusion: 1. We found that from 2002 to 2014, we found that COM was the most common stone component, COD and MAP stones had an increasing trend, while CaP stones, UA stones and cystine stones had a decreasing trend, and the urinary calculus components were in different sex and age. There is a significant difference between the groups of.2. Metformin can effectively alleviate the lipid peroxidation induced by oxalic acid and the resulting renal tubular epithelial cell damage. More importantly, this study was the first to confirm the effect of metformin in the treatment of calcium oxalate kidney stones and in vitro and in vivo animal models. It is confirmed that metformin can inhibit the renal crystallization of.3 by antioxidation mechanism. This study also confirmed that metformin can effectively alleviate the overexpression of OPN and MCP-1 in renal tubular epithelial cells induced by oxalate in the process of calcium oxalate kidney stones. Metformin may be an effective drug for the treatment and prevention of calcium oxalate nephrolithiasis, and this may greatly benefit the patients with primary high oxalate and recurrent calcium oxalate kidney stones.
【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R692.4

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