联合使用二甲双胍与2-脱氧葡萄糖抑制小型猪多囊肾病进展的研究

发布时间：2018-06-27 08:21

本文选题：多囊肾病 + 二甲双胍　；参考：《中国人民解放军医学院》2017年硕士论文

【摘要】：目的:常染色体显性多囊肾病(ADPKD)是最常见遗传性肾病,目前尚无有效的预防及治疗药物。已发现多囊肾细胞与肿瘤细胞类似,主要依赖糖酵解产生ATP。2-Deoxy-D-glucose(2DG)是糖酵解阻断剂,二甲双胍是AMPK激活剂,目前两者已用于肿瘤治疗。小型猪与啮齿类动物相比,在代谢、生理等方面与人更加相似,是研究ADPKD机制的一种理想大动物模型。本研究首次从能量代谢的角度入手,联合使用抗代谢药物二甲双胍和2-脱氧葡萄糖长期治疗缓慢进展性ADPKD小型猪模型,观察了两种药物单独或者联合对ADPKD小型猪的治疗效果以及对肾脏组织能量代谢、细胞增殖的影响。方法:(1)随机挑选5-6月龄的通过锌指核酶(ZFN)技术敲除PKD1基因的中国实验用小型猪作为多囊肾模型组(PKD),选取相同月龄相同品系的野生型小型猪作为正常对照组(CON)。喂养20个月,按规定日期麻醉后取血、尿标本,然后处死动物,制备肾脏组织标本。观察血生化、尿蛋白、肾脏病理改变、细胞周期相关蛋白、增殖、代谢的变化情况。(2)将PKD1基因敲除的小型猪,随机分为对照组,二甲双胍干预组,2DG干预组,二甲双胍和2DG联合干预组。第一批小型猪饲养10个月后,按规定日期麻醉后取血、尿标本,然后处死动物,制备肾脏组织标本。为了观察长期疗效,第二批小型猪饲养至20个月时,麻醉后通过局部手术的方式取部分肾组织,小型猪苏醒后继续饲养。观察指标同上。结果:(1)、结果发现在ADPKD小型猪肾脏中糖酵解水平明显升高。mTOR信号通路和ERK信号通路明显激活。(2)、治疗10个月后处死部分动物,肾脏外观和剖面图以及CT所测囊肿指数结果均显示:各药物干预组与对照组相比有明显差异;其中联合用药组差异最显著;但各组血、尿生化结果水平无明显改变。治疗20个月后,生化结果显示,各用药组与对照组相比血清肌酐及尿蛋白/尿肌酐比值明显降低。为了长期观察治疗效果,采用手术的方式取肾脏组织,结果显示:与对照组相比,药物干预组小型猪肾脏组织中增殖信号通路mTOR以及ERK的关键分子表达水平有不同程度的下降,其中以联合用药组下降效果最明显。结论:本研究显示ADPKD小型猪肾脏中糖酵解水平明显和增殖明显增高。用二甲双胍和2DG药物联合干预可明显改善ADPKD小型猪肾功能以及延缓肾脏囊泡的进展。这为2DG和二甲双胍治疗ADPKD提供了强有力的实验依据。
[Abstract]:Objective: autosomal dominant polycystic kidney disease (ADPKD) is the most common hereditary nephropathy. It has been found that polycystic kidney cells are similar to tumor cells and mainly rely on glycolysis to produce ATP.2-Deoxy-D-glucose (2DG) as glycolytic blockers and metformin as AMPK activators. Compared with rodents, miniature pigs are more similar to humans in metabolism, physiology and so on. It is an ideal animal model to study ADPKD mechanism. In this study, energy metabolism was used for the first time to treat slowly progressive ADPKD miniature pig model with the combination of metformin and 2-deoxyglucose for a long time. The effects of two drugs alone or in combination on ADPKD mini-pigs and on renal energy metabolism and cell proliferation were observed. Methods: (1) Chinese miniature pigs (PKD) with zinc finger ribozyme (ZFN) knockout of PKD1 gene were randomly selected as polycystic kidney model group (PKD) and wild miniature pigs of the same age and the same age as normal control group (Con). After 20 months of feeding, blood and urine samples were collected after anesthesia, then animals were killed and kidney tissue specimens were prepared. The changes of blood biochemistry, urine protein, renal pathological changes, cell cycle related protein, proliferation and metabolism were observed. (2) miniature pigs with PKD1 gene knockout were randomly divided into control group and metformin intervention group. Metformin and 2DG combined intervention group. The first miniature pigs were fed for 10 months. Blood and urine samples were collected after anesthesia according to the prescribed date. Then animals were killed and kidney tissue specimens were prepared. In order to observe the long-term curative effect, the second batch of miniature pigs were fed for 20 months. After anesthesia, partial renal tissues were taken out by local operation, and the small pigs were reared after waking up. Observation indicators ibid. Results: (1) the level of glycolysis in the kidney of ADPKD miniature pigs was significantly increased. The signal pathway of mTOR and ERK were activated obviously. (2) after 10 months of treatment, some animals were killed. The renal appearance and profile as well as the cysts index measured by CT showed that there were significant differences between each drug intervention group and the control group, and the most significant difference was found in the combined treatment group, but there was no significant change in the blood and urine biochemical results in each group. After 20 months of treatment, the biochemical results showed that the serum creatinine and urinary creatinine / urinary creatinine ratio were significantly lower in each treatment group than those in the control group. In order to observe the therapeutic effect for a long time, the renal tissue was removed by operation. The results showed that the expression levels of mTOR and ERK key molecules in the renal tissue of the drug intervention group were lower than those in the control group. Among them, the decrease effect of combined drug group was the most obvious. Conclusion: this study showed that the glycolysis level and proliferation of ADPKD miniature pig kidney were significantly increased. Combined intervention of metformin and 2DG could significantly improve renal function and delay the progression of renal vesicles in ADPKD miniature pigs. This provides a powerful experimental basis for the treatment of ADPKD with 2 DG and metformin.
【学位授予单位】：中国人民解放军医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R692

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