本文選題：產(chǎn)甲酸草酸桿菌　切入點：尿石癥　出處：《第二軍醫(yī)大學》2017年碩士論文　論文類型：學位論文

【摘要】：研究背景及目的目前腎結(jié)石是世界排名第三的泌尿系統(tǒng)疾病,由于人口老齡化、全球氣候變暖、生活及飲食方式改變、肥胖流行以及更加精確的診斷手段等原因,腎結(jié)石患病率將進一步提高。過去,人們往往認為腎結(jié)石是一種急性病,但越來越多的證據(jù)顯示它亦是一種慢性系統(tǒng)性疾病,最終可能引起終末期腎臟病。晚近有研究發(fā)現(xiàn)腎結(jié)石患者心血管疾病風險增高。草酸鈣結(jié)石是腎結(jié)石最常見的類型,占所有結(jié)石的70%-80%。尿液中草酸濃度升高是草酸鈣結(jié)石形成的重要因素,過度飽和的草酸和鈣結(jié)合形成草酸鈣結(jié)晶,足夠多的草酸鈣結(jié)晶聚合形成草酸鈣結(jié)石。尿草酸含量受到飲食中草酸、鈣和維生素D攝入以及內(nèi)源性草酸產(chǎn)生的影響。20%~53%的尿草酸來自飲食中的草酸攝入,即外源性草酸。內(nèi)源性草酸是肝臟降解甘氨酸、乙醛酸等的終產(chǎn)物,人體主要通過三種渠道排泄草酸:5%~10%分泌到腸腔,與鈣離子結(jié)合形成不溶性草酸鈣隨糞便排出體外或被腸道微生物降解,90%~95%經(jīng)腎臟隨尿液排出,因此增加腸道中草酸降解和排泄可能是預防和治療草酸鈣結(jié)石的有效方法之一。1985年科學家首次發(fā)現(xiàn)腸道產(chǎn)甲酸草酸桿菌可降解草酸,人體腸道產(chǎn)甲酸草酸桿菌密度差異很大,從完全缺乏到108/g腸內(nèi)容物都普遍存在,高含量的產(chǎn)甲酸草酸桿菌每日可降解0.5-1g草酸,而絕大多數(shù)草酸鈣腎結(jié)石患者的腸道中缺乏這種菌,因此補充這種益生菌可大大降低草酸鈣腎結(jié)石的發(fā)生及復發(fā)風險。目前關(guān)于產(chǎn)甲酸草酸桿菌的研究大部分集中于國外,國內(nèi)相關(guān)研究甚少,本研究的目的是從健康人腸道中篩選出產(chǎn)甲酸草酸桿菌,研究其預防草酸鈣結(jié)石的作用,為國內(nèi)相關(guān)藥物研發(fā)提供可靠依據(jù)。研究方法1、從健康人新鮮糞便中篩選產(chǎn)甲酸草酸桿菌,經(jīng)分離、培養(yǎng)、純化、鑒定得到產(chǎn)甲酸草酸桿菌菌株,將菌株擴增至不同濃度(106cfu/m L、107cfu/m L、108cfu/m L)。2、在雄性SD大鼠飲水中添加0.8%乙二醇建立草酸鈣結(jié)石模型。3、每日以1m L活菌干預草酸鈣結(jié)石動物模型,定期稱重并收集大鼠血樣及尿樣,檢測血鈣、血鎂、血磷、BUN、Scr、尿草酸、尿鈣、尿鎂、尿磷變化,4周后麻醉大鼠,取其腎臟,橫切片并作HE染色,偏光鏡下觀察腎臟結(jié)晶含量,作Yasue染色觀察草酸鹽沉積。研究結(jié)果1、經(jīng)16s rRNA序列測定,我們從健康人新鮮糞便中分離出的產(chǎn)甲酸草酸桿菌與已知ATCC35274相似度為100%,由此判定所獲得菌種為產(chǎn)甲酸草酸桿菌。2、飲水中添加0.8%EG建立草酸鈣結(jié)石模型后大鼠24小時尿草酸排泄量顯著升高,濃度梯度產(chǎn)甲酸草酸桿菌干預草酸鈣結(jié)石模型4周,106cfu、107cfu治療組大鼠24小時尿草酸排出量與單純造模組差異無統(tǒng)計學意義,而108cfu治療組在第一周末24小時尿草酸排泄量顯著低于造模組,且持續(xù)至第四周末。3、干預4周后,106cfu、107cfu組與造模組草酸評分差異無統(tǒng)計學意義,108cfu組腎臟草酸評分顯著低于造模組。4、造模組大鼠體重、Scr、BUN較對照組相比差異無統(tǒng)計學意義,說明草酸鈣結(jié)石模型的腎功能未受到影響。與對照組及造模組相比,三個治療組大鼠體重及腎功能差異無統(tǒng)計學意義,說明產(chǎn)甲酸草酸桿菌耐受性好。研究結(jié)論本實驗已篩選出人源產(chǎn)甲酸草酸桿菌,并將菌種保藏于中國典型培養(yǎng)物保藏中心,且已在Genebank申請序列,目前培養(yǎng)最高濃度為107cfu/ml,還需要進一步對菌種進行優(yōu)化,提高發(fā)酵濃度,縮短發(fā)酵時間;每日給予108cfu產(chǎn)甲酸草酸桿菌可安全、有效降低大鼠尿草酸排泄量,有效預防腎臟草酸鈣結(jié)晶形成從而抑制結(jié)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.

【學位授予單位】：第二軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R692.4

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相關(guān)期刊論文 前1條

1 陳志強,郭輝,葉章群,朱旭慧,余建華,曾令啟;人腸道產(chǎn)甲酸草酸桿菌的分離培養(yǎng)[J];中華實驗外科雜志;2005年11期

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本文編號：1629798