产甲酸草酸杆菌预防草酸钙肾结石的作用研究

发布时间：2018-03-18 13:34

本文选题：产甲酸草酸杆菌　切入点：尿石症　出处：《第二军医大学》2017年硕士论文　论文类型：学位论文

【摘要】：研究背景及目的目前肾结石是世界排名第三的泌尿系统疾病,由于人口老龄化、全球气候变暖、生活及饮食方式改变、肥胖流行以及更加精确的诊断手段等原因,肾结石患病率将进一步提高。过去,人们往往认为肾结石是一种急性病,但越来越多的证据显示它亦是一种慢性系统性疾病,最终可能引起终末期肾脏病。晚近有研究发现肾结石患者心血管疾病风险增高。草酸钙结石是肾结石最常见的类型,占所有结石的70%-80%。尿液中草酸浓度升高是草酸钙结石形成的重要因素,过度饱和的草酸和钙结合形成草酸钙结晶,足够多的草酸钙结晶聚合形成草酸钙结石。尿草酸含量受到饮食中草酸、钙和维生素D摄入以及内源性草酸产生的影响。20%~53%的尿草酸来自饮食中的草酸摄入,即外源性草酸。内源性草酸是肝脏降解甘氨酸、乙醛酸等的终产物,人体主要通过三种渠道排泄草酸:5%~10%分泌到肠腔,与钙离子结合形成不溶性草酸钙随粪便排出体外或被肠道微生物降解,90%~95%经肾脏随尿液排出,因此增加肠道中草酸降解和排泄可能是预防和治疗草酸钙结石的有效方法之一。1985年科学家首次发现肠道产甲酸草酸杆菌可降解草酸,人体肠道产甲酸草酸杆菌密度差异很大,从完全缺乏到108/g肠内容物都普遍存在,高含量的产甲酸草酸杆菌每日可降解0.5-1g草酸,而绝大多数草酸钙肾结石患者的肠道中缺乏这种菌,因此补充这种益生菌可大大降低草酸钙肾结石的发生及复发风险。目前关于产甲酸草酸杆菌的研究大部分集中于国外,国内相关研究甚少,本研究的目的是从健康人肠道中筛选出产甲酸草酸杆菌,研究其预防草酸钙结石的作用,为国内相关药物研发提供可靠依据。研究方法1、从健康人新鲜粪便中筛选产甲酸草酸杆菌,经分离、培养、纯化、鉴定得到产甲酸草酸杆菌菌株,将菌株扩增至不同浓度(106cfu/m L、107cfu/m L、108cfu/m L)。2、在雄性SD大鼠饮水中添加0.8%乙二醇建立草酸钙结石模型。3、每日以1m L活菌干预草酸钙结石动物模型,定期称重并收集大鼠血样及尿样,检测血钙、血镁、血磷、BUN、Scr、尿草酸、尿钙、尿镁、尿磷变化,4周后麻醉大鼠,取其肾脏,横切片并作HE染色,偏光镜下观察肾脏结晶含量,作Yasue染色观察草酸盐沉积。研究结果1、经16s rRNA序列测定,我们从健康人新鲜粪便中分离出的产甲酸草酸杆菌与已知ATCC35274相似度为100%,由此判定所获得菌种为产甲酸草酸杆菌。2、饮水中添加0.8%EG建立草酸钙结石模型后大鼠24小时尿草酸排泄量显著升高,浓度梯度产甲酸草酸杆菌干预草酸钙结石模型4周,106cfu、107cfu治疗组大鼠24小时尿草酸排出量与单纯造模组差异无统计学意义,而108cfu治疗组在第一周末24小时尿草酸排泄量显著低于造模组,且持续至第四周末。3、干预4周后,106cfu、107cfu组与造模组草酸评分差异无统计学意义,108cfu组肾脏草酸评分显著低于造模组。4、造模组大鼠体重、Scr、BUN较对照组相比差异无统计学意义,说明草酸钙结石模型的肾功能未受到影响。与对照组及造模组相比,三个治疗组大鼠体重及肾功能差异无统计学意义,说明产甲酸草酸杆菌耐受性好。研究结论本实验已筛选出人源产甲酸草酸杆菌,并将菌种保藏于中国典型培养物保藏中心,且已在Genebank申请序列,目前培养最高浓度为107cfu/ml,还需要进一步对菌种进行优化,提高发酵浓度,缩短发酵时间;每日给予108cfu产甲酸草酸杆菌可安全、有效降低大鼠尿草酸排泄量,有效预防肾脏草酸钙结晶形成从而抑制结石形成。
[Abstract]:Background and purpose the kidney stone is ranked third in the world of urinary system diseases, due to an aging population, global warming, changes to lifestyle and diet, obesity epidemic and more accurate diagnostic methods, will further increase the rate of prevalence of kidney stones. The past, people tend to think that the kidney stone is a kind of acute disease. But more and more evidences show that it is a chronic systemic disease, may eventually cause of end-stage renal disease. Recent studies have found an increased risk of cardiovascular disease in patients with kidney stones. Calcium oxalate is the most common type of kidney stones, accounting for 70%-80%. of all urine oxalic acid concentration in stone increased is an important factor in the formation of calcium oxalate calculus the combination of over saturated oxalic acid and calcium to form calcium oxalate, calcium oxalate stone formation polymerization enough calcium oxalate. Oxalate content by dietary oxalate, Calcium and vitamin D intake and urinary oxalate effects of endogenous.20%~53% produced from oxalic acid in the diet intake of oxalic acid, namely Exogenous Oxalic acid. The endogenous oxalic acid is the end product of the liver glycine, glyoxalic acid, the human body mainly through three channels: 5%~10% points to urinary oxalate excretion of intestinal cavity combined with calcium to form insoluble of calcium oxalate excreted with the feces or by intestinal microbial degradation, 90%~95% is excreted in the urine by the kidneys, thereby increasing the degradation of oxalic acid and excretion in the gut may be one of the effective methods for prevention and treatment of calcium oxalate stone.1985 scientists first discovered the intestinal oxalobacter formigenes degradable oxalic acid, human intestinal oxalobacter formigenes density difference from the complete lack of 108/g, intestinal contents are widespread, high content of oxalobacter formigenes daily degradable 0.5-1g oxalic acid, while the vast majority of calcium oxalate kidney Patients with intestinal stone lack this bacterium, therefore this probiotics can greatly reduce the risk of the occurrence and recurrence of calcium oxalate kidney stones. The current research on the oxalobacter formigenes most concentrated in foreign countries, domestic related research is very little, the purpose of this study is to screen out formigenes from healthy human gut, study the prevention of oxalic acid the role of calcium stones, and provide a reliable basis for the domestic research and development of the related drugs. Methods 1 healthy people from the fresh feces were oxalobacter formigenes, after separation, purification, culture, identification of oxalobacter formigenes strains, the strain amplified to different concentrations (106cfu/m L, 107cfu/m L, 108cfu/m L.2, add) 0.8% ethylene glycol establish calcium oxalate stone model.3 in drinking water in male SD rats by 1m L, the daily live bacteria intervention of calcium oxalate stone animal model, weighed and collected blood samples and urine samples of rats with regular inspection Measurement of serum calcium, serum magnesium, phosphorus, BUN, Scr, urinary oxalate, urinary calcium, urinary magnesium, urinary phosphorus changes of rats after 4 weeks of anesthesia, kidneys, and cross sections for HE staining, kidney crystalline content was observed under a polarized light microscope, Yasue staining was observed in oxalate deposition. The results by 1. 16S rRNA sequence determination of oxalic acid, formic acid production were isolated from healthy human faeces bacillus with known ATCC35274 100% similarity result obtained by strain.2 of oxalobacter formigenes in drinking water, add 0.8%EG of calcium oxalate stone model rats after 24 hours urinary oxalate excretion increased significantly, the concentration gradient of formic acid production oxalobacter intervention of calcium oxalate stone model for 4 weeks, 106cfu, 107cfu treated rats 24 hours urinary oxalate excretion and simple model of the difference was not statistically significant, while the 108cfu group in the first weekend of 24 hours urinary oxalate excretion was significantly lower than that of model group, and continued to fourth .3 weekend, 4 weeks after the intervention, 106cfu, no statistical significance 107cfu group and model group 108cfu score between oxalic acid, oxalate kidney score was significantly lower than that of model.4, made in weight, model rats Scr, BUN compared to the control group, the difference was not statistically significant, explain kidney function model of calcium oxalate stones were not affected. The control group and model group compared with no significant three treatment body weight and renal function in rats showed differences in oxalobacter formigenes were well tolerated. Conclusion this experiment has been selected from oxalobacter formigenes, and the strain is preserved in China Center for type culture collection, and has applied for sequence in Genebank at present, training for the highest concentration of 107cfu/ml, also need to further optimize the bacteria, improve the fermentation concentration, shorten the fermentation time; a daily dose of 108cfu of oxalobacter formigenes safe, effectively reduce rat urinary oxalate excretion, It effectively prevents the crystallization of calcium oxalate in the kidney and inhibits the formation of stones.

【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R692.4

【参考文献】