三聚氰胺及三聚氰酸诱导大鼠肾脏损害及结石的研究

发布时间：2018-01-03 17:02

  本文关键词：三聚氰胺及三聚氰酸诱导大鼠肾脏损害及结石的研究　出处：《南方医科大学》2010年硕士论文　论文类型：学位论文

  更多相关文章： 三聚氰胺 三聚氰酸 三聚氰胺氰尿酸盐 尿酸结石 急性肾功能衰竭

【摘要】： 研究背景 研究显示2007年3月美国大量宠物中毒发生急性肾衰事件,是由于宠物食物中混有8.4%的三聚氰胺和5.3%三聚氰酸；而导致我国2008年9月因服用“问题奶粉”出现近30万婴幼儿发生泌尿系结石及泌尿系异常的原因则是“问题奶粉”中含有大量浓度不等的三聚氰胺。研究二者诱导肾脏损害及泌尿系结石的病理过程和发病机制,寻找预防和治疗此类肾脏损害和结石的有效方法,是目前国内外专家们急需积极研究的热点方向。 三聚氰胺(MA,英文名Melamine),简称三胺,常用分子式为C3N6H6,是一种三嗪类含氮杂环有机化合物。外观为粉末状纯白色单斜棱晶体,微溶于水,低毒,大鼠口服的半数致死量大于3克/公斤体重。主要用于合成三聚氰胺甲醛树脂,以制作复合板、塑料、涂料、商用过滤器、胶水或粘合剂以及餐具和厨具。在三聚氰胺制造过程中产生的杂质可能会形成氰尿酸、三聚氰酸二酰胺和三聚氰酸一酰胺等同系物。如果三聚氰胺未能完全代谢为氨和二氧化碳,经细菌代谢,也可能会形成其同系物。 三聚氰酸(CA,英文名Cyanuric acid等)；又名氰尿酸,异氰脲酸等,是由尿素聚合而得的白色结晶,分子式：C3H3N3O3,亦是一种重要的三嗪类含氮杂环有机化合物。微溶于水,基本无毒,大鼠LD50为7.7g/kg。用于有机合成氰尿酸一甲醛树脂、涂料、粘合剂、农药除草剂、金属氰化缓蚀剂、高分子材料改性剂和用于药物卤三羟嗪的生产等。 而最近的研究则发现被包括美国在内的许多国家广泛用来消毒食品生产设备、蔬菜和水果的三氯甲烷(Trichloromelamine),经分解能生成三聚氰胺；而另外一种叫环丙氨嗪(cyromazine)的农药也可以残留在蔬菜和水果的表面进而分解成三聚氰胺。一种叫二氯异氰尿酸(dichloroisocyanurates)的消毒剂则被用来作为饮用水和游泳池水的消毒剂,而其可以代谢生成三聚氰酸。故三聚氰胺和三聚氰酸与人类生活已经密切接触。 既往研究显示三聚氰胺和三聚氰酸在哺乳动物体内属于惰性代谢,即在体内不会被转化,亦不会蓄积体内,均以原型从尿液中排出。其中三聚氰胺在体内的半衰期约为2.7h,三聚氰酸为2.5h,因此泌尿系统容易成为三聚氰胺与三聚氰酸损害的靶器官。国外的动物实验显示长期单独大剂量摄入三聚氰胺或三聚氰酸导致膀胱癌、膀胱结石、肾小管上皮增生、扩张及肾纤维化等。而2008年Dobson等人的研究证实了三聚氰胺和三聚氰酸同时混合摄入导致大鼠急性肾衰竭而死亡,且病理解剖提示肾小管堵塞了大量结晶,同时发现其他器官未受明显影响,并通过检测发现肾组织中的结晶是三聚氰胺和三聚氰酸1：1结合的晶体。另外Dobson等人的研究还证明了单独摄入三聚氰胺或三聚氰酸并未对大鼠肾脏造成损害,而且三聚氰胺或三聚氰酸在体内所能达到的浓度限制内是不会对肾脏细胞造成毒性作用的。目前同系物三聚氰酸二酰胺和三聚氰酸一酰胺尚无致肾损害及结石的数据。 由于三聚氰胺含氮量较高,因而被非法添加到宠物饲料、牛奶以及其它蛋白制品中以虚假提高蛋白含量。然而纯度不高的三聚氰胺往往混杂有三聚氰酸,或者在食品加工过程中三聚氰胺可以少量转化成三聚氰酸。我国“问题奶粉”事件后质检总局随后在全国开展了婴幼儿奶粉专项检查,其中在某品牌奶粉中检测出三聚氰胺含量高达2563mg/kg,但未报道检测出三聚氰酸。此外,北京大学一附院的研究发现暴露在三聚氰胺的患儿泌尿系结石发病率比正常婴幼儿高出7倍,北京儿童医院分析出“问题奶粉”患儿结石成分为“三聚氰胺和尿酸”,所以目前国内一直认为是三聚氰胺与尿酸造成婴幼儿发生肾结石。 急性肾衰(ARF)的发病机制主要分为肾前性、肾性和肾后性,而三聚氰胺和三聚氰酸被证明无直接肾毒性,其对肾脏损害的主要原因可能是肾后性梗阻引起的ARF。泌尿系结石的种类和发病机制十分复杂,而目前研究证明与三聚氰胺相关的结石很可能与尿酸一类结石相关,这可能与三嗪类化合物影响体内代谢,以及其分子结构更易诱导尿酸的趋向附生而形成结石有关,故需要研究三聚氰胺和三聚氰酸对尿酸排泄量、尿PH值、尿量等诱发尿酸结石的关键因素的影响。 “问题奶粉”对泌尿系损害机制尚未完全明确,基于受检测的问题奶粉中含三聚氰胺最高剂量为2563mg/kg,再根据婴儿最小平均体重(6kg)、平均每日摄入奶粉量(150g)及大鼠与人体药理换算公式(约6.3：1),我们计算出复制大鼠模型每天应摄入最大三聚氰胺剂量约为403.7 mg/kg。同时参考国外的动物实验剂量,设计了本实验给药剂量进行研究。本实验采用三聚氰胺和三聚氰酸单独及联合灌胃给药方法,建立大鼠肾脏损害模型进行研究,旨在探明三聚氰胺及其同系物三聚氰酸对肾脏损害和诱发结石的机制,为进一步寻求防治方法提供实验依据。 研究目的： 1、探讨单独的三聚氰胺或三聚氰酸对大鼠肾脏损害及诱发泌尿系结石的机制,并分析结石晶体成分。 2、探讨三聚氰胺和三聚氰酸的联合效应损害大鼠肾脏及诱发泌尿系结石的机制,并分析结石晶体成分。 3、通过大鼠的实验研究来探讨“问题奶粉”致婴幼儿泌尿系结石的可能机制。 研究方法： 1、制作模型和分组 将60只SD大鼠随机分成A组(空白对照组)、B组(三聚氰胺400mg·kg-1·d-1)、C组(三聚氰酸400mg·kg-1·d-1)、D组(三聚氰胺400mg·kg-1·d-1 +三聚氰酸400mg·kg-1·d-1)、E组(三聚氰胺30mg·kg-1·d-1+三聚氰酸30mg·kg-1·d-1),每组12只,通过灌胃给药。 2、指标的收集和检测 用代谢笼收集前3天24小时尿液,进行24小时尿量、PH值和24小时尿酸排泄量测量及尿沉渣涂片检查；分别于第4天、第31天灌胃前每组采集6只大鼠的血液标本检测血肌酐、尿素氮、尿酸；取双侧肾脏用外科显微镜观察后,制作快速冰冻切片及石蜡切片(HE染色)观察肾脏病理改变及晶体形成。肾脏冰冻组织切成5μm薄片后,用红外显微光谱仪检测晶体成分。 3、采用SPSS 13.0统计软件进行数据分析,数据以x±s表示。尿液数据和血液数据分别采用重复测量方差分析和析因方差分析。当P0.05时,差异有统计学意义。 结果： 1、尿液和血液生化结果 与A组比较,B组和C组大鼠的24h尿尿量、24h尿酸排泄量均明显增多,差异均有统计学意义(P0.05)；而D、E组尿量明显减少、PH值降低、24h尿酸排泄量明显减少,差异均有统计学意义(P0.05),其中D组变化比E组更为明显,差异有统计学意义(P0.05)。在相应时间点各组与A组比较,B组和C组的血肌酐、血尿酸及血尿素氮均升高,但在第4天时候仅血尿素氮差异有统计学意义(P0.05),而第31天血尿素氮及血尿酸差异均有统计学意义(P0.05)；D组在第4天的时候各项指标均显著升高,差异有统计学意义(P0.05)；E组第4天血肌酐及血尿素氮明显升高,差异有统计学意义(P0.05),第31天则各项指标明显升高,差异均有统计学意义(P0.05)。其中D组比E组变化更为明显,差异有统计学意义(P0.05),同时E组各项指标第31天比第4天显著升高,差异有统计学意义(P0.05)。(见表2) 2、尿沉渣涂片 从尿沉渣涂片中可见：A组尿液无明显晶体；B组可见疑似三聚氰胺原型的晶体；C组无明显晶体,但有少许细沙样小颗粒沉淀物；D组及E组均可见淡黄色圆形特异性晶体。 3、肾脏病理改变 大体标本：A组大鼠肾脏包膜光滑暗红色,肾脏解剖观察无明显病理改变。B、C组大鼠肾脏皮髓结构清晰,肾盏及肾盂良好,未见结石晶体沉积。D组大鼠肾脏明显肿大,外观呈灰黄色,包膜有许多散在黄色斑点,肾脏皮质变薄,肾盏、肾盂结构破坏,肾内布满淡黄色结石晶体。E组第4天皮髓结构尚清晰,可见不少淡黄色结石晶体沉积；第31天E组大鼠肾脏剖面呈红褐色,皮髓变模糊,大量淡黄色颗粒沉积。各组大鼠膀胱均未见观察到结石。 肾组织切片：A组大鼠未见明显病理改变。B组及C组肾组织石蜡切片与冰冻切片在第4天的时候均未见结晶及明显病理改变；第31天亦未见晶体,仅见少量肾小管轻度扩张、上皮细胞肿胀和少量管型形成等。D组与E组肾组织石蜡切片与冰冻切片均可见淡黄色、呈放射状的同心圆形晶体,主要集聚于肾小管,也见于集合管、间质及被膜下,偶见于肾小球囊中,大多晶体以不规则碎片出现于肾小管中。此外,肾小管浊肿、上皮受损,并有各种管型形成,大量炎性细胞在肾小球囊腔、肾小管及间质中；E组第31天还可看到许多纤维细胞充满间质。此外第4’天D组切片单倍视野下结石晶体数量和体积,以及组织病理变化都大于E组；而第31天E组结石晶体数量、体积以及组织病理变化都比其在第4天观察到的要大。 4、肾组织晶体成分分析结果 对混合组大鼠肾脏出现的各种形态结石晶体进行红外显微光谱分析,得到基本一致的红外光谱图；根据专业分析,及查阅相关文献可知混合药物组大鼠在肾脏生成的晶体即为三聚氰胺氰尿酸盐晶体。 结论： 1、三聚氰胺和三聚氰酸同时混合进入体内,会在肾脏形成一种不溶于水的三聚氰胺氰尿酸盐晶体,此晶体能堵塞肾小管造成肾脏损害甚至急性肾衰竭,并可以作为结石核心与尿酸等成分一起诱发泌尿系结石； 2、三聚氰胺与其同系物三聚氰酸对肾脏的损害较轻,机制相似；单独摄入三聚氰胺或三聚氰酸,大鼠肾脏无结石晶体形成,但长期摄入会对肾功能造成损害,其原因为长期的渗透性利尿导致物理性损伤肾组织； 3、三聚氰胺与三聚氰酸的联合效应比它们的单独效应对肾脏的损害作用更大,更容易诱发泌尿系尿酸结石；其联合效应与联合药物的浓度和暴露时间成正相关。 4、“问题奶粉”致婴幼儿泌尿系结石的机制较复杂,但奶粉中同时存在三聚氰胺和三聚氰酸可能是主要诱发因素。
[Abstract]:Research background
Research shows that the United States in March 2007 a large number of pets poisoning events in acute renal failure is due to the pet food mixed with 8.4% melamine and 5.3% cyanuric acid; in September 2008 in China by taking the "milk" causes abnormal urinary calculi and urinary tract in 300 thousand infants and young children is "milk" contains a large number of varying concentrations melamine. The pathological process and mechanism of two induced renal damage and urinary calculi, effective method for the prevention and treatment of the renal damage and stones, is a hot topic at home and abroad experts to explore actively.
Melamine (MA, English name Melamine), referred to as three amine, molecular formula C3N6H6, is one of three triazine heterocyclic organic compounds. The appearance of pure white powder monoclinic crystal, slightly soluble in water, low toxicity, rat oral lethal dose greater than 3 g / kg body weight is mainly used. Synthesis of melamine formaldehyde resin, to produce composite panels, plastic, paint, glue or adhesive and commercial filters, tableware and kitchenware. The impurities produced in the manufacturing process of melamine may form cyanuric acid, cyanuric acid amide and two ammelide equivalent series. If the melamine is not completely metabolized to ammonia and carbon dioxide by bacteria metabolism, may also form its homologues.
Cyanuric acid (CA, English Cyanuric acid); also known as cyanuric acid, isocyanuric acid, is a white crystal, derived from urea polymer molecular formula: C3H3N3O3, is also an important class of three triazine heterocyclic organic compounds. Slightly soluble in water, basic non-toxic, rat LD50 7.7g/kg. used for organic synthesis of cyanuric acid formaldehyde resin, coating, adhesive, herbicide, metal cyanide inhibitor, polymer modifiers and drugs for halogen three hydroxyzine production.
A recent study found that many countries including the United States, widely used for disinfection of food production equipment, fruit and vegetables (Trichloromelamine), chloroform into melamine; and another called cyromazine (cyromazine) pesticide can also be left on the surface of fruits and vegetables and decomposed into melamine. Called two chloro isocyanuric acid (dichloroisocyanurates) was used as a disinfectant for drinking water and swimming pool water disinfectant, and its metabolism can produce cyanuric acid. Therefore, melamine and cyanuric acid and human life have close contact.
Previous studies have shown that melamine and cyanuric acid are inert metabolism in mammals, which transformed the body will not, will not accumulate in the body, discharge from the urine in the prototype. The half-life of melamine in vivo is about 2.7h, cyanuric acid, 2.5h, urinary organ so easily become the melamine and cyanuric acid damage. Foreign animal experiments showed that long-term high-dose single intake of melamine or cyanuric acid lead to bladder cancer, bladder stones, renal tubular epithelial hyperplasia, expansion and renal fibrosis. In 2008, Dobson et al confirmed that melamine and cyanuric acid and mixed ingestion rats with acute renal failure and death, and the pathological anatomy of renal tubular plug tips a large number of crystal, also found that other organs were not significantly affected, and through the detection of crystallization in renal tissue is melamine and three Polycyanate with 1:1 crystal. In addition Dobson et al also demonstrated that a separate intake of melamine or cyanuric acid did not cause damage to the kidney of rats, and limit the concentration of melamine or cyanuric acid in the body can reach the inside will not cause toxic effects on renal cells. At present, cyanuric acid and cyanuric acid, an amide two there is no amide induced kidney damage and the stones of the data.
Due to high nitrogen content of melamine, which was illegally added to pet food, milk and other protein products to increase protein content. However, no false high purity melamine is often mixed with melamine or cyanuric acid in food processing can be converted into a small amount of cyanuric acid. China's "milk" incident and quality inspection administration in the country to carry out a special inspection of infant formula, in which a certain brand of milk powder detected melamine content of up to 2563mg/kg, but did not report detected cyanuric acid. In addition, a Peking University Study Institute found that exposure to the children with urinary calculi incidence rate is 7 times higher than the normal infant melamine, Beijing Children's Hospital of "milk" the children as "melamine and uric acid stones", so at home has been considered to be made of melamine and uric acid Nephrolithiasis occurs in infants and young children.
Acute renal failure (ARF) mechanism is divided into pre renal, renal and post renal, and melamine and cyanuric acid proved no direct renal toxicity, the main reason for renal damage may be the type and the pathogenesis of ARF. urinary calculi after renal obstruction caused by ten points and the current complex. Study on the calculus of proof and melamine related may be related to a class of uric acid stones, which may affect the metabolism of three triazine compounds, and its molecular structure is more easily induced and the formation of the trend of epiphytic uric acid stones, it needs to study the melamine and cyanuric acid on uric acid excretion, urine pH value, the key factors affecting the urine the amount of induced uric acid calculi.
Mechanism of "milk" on the urinary system damage is not completely clear, by the problem of detecting melamine in milk based on the highest dose of 2563mg/kg, according to the average weight of the smallest babies (6kg), the average daily intake of milk volume (150g) and rat and human pharmacological conversion formula (about 6.3:1), we calculate the day the rat model of maximum intake of melamine dose is about 403.7 mg/kg. at the same time reference dose of animal experiments abroad, designed the dose for this experiment. This experiment uses melamine and cyanuric acid alone or in combination with gavage method, establish rat renal damage model is studied, aiming to explore the melamine and cyanuric acid on kidney the mechanism of damage and induced calculi, provide experimental basis for further seeking prevention and treatment methods.
The purpose of the study is:
1, the mechanism of single melamine or cyanuric acid on kidney damage and urinary calculi induced in rats was investigated and the crystal composition of the stone was analyzed.
2, the mechanism of combined effects of melamine and cyanuric acid on kidney and urinary calculi in rats was investigated and the crystal composition of the stone was analyzed.
3, through the experimental study of rats, the possible mechanism of "problem milk powder" to infantile urinary calculi was discussed.
Research methods:
1, making models and grouping
60 SD rats were randomly divided into group A (control group), group B (400mg - kg-1 - D-1 melamine), C group (400mg - kg-1 - D-1 cyanuric acid), D group (400mg - kg-1 - D-1 + melamine cyanuric acid 400mg - kg-1 - D-1), group E (melamine 30mg kg-1 - d-1+ - kg-1 - 30mg cyanuric acid D-1), with 12 rats in each group by intragastric administration.
2, the collection and detection of indicators
In 3 days, 24 hours urine metabolic cages were collected before, 24 hours and 24 hours urine volume, pH measurement of uric acid excretion and urinary sediment smears; on the fourth day, thirty-first days with creatinine, blood samples collected before gastric blood each 6 rats in blood urea nitrogen, uric acid; the bilateral kidneys were observed by surgical microscope, making rapid frozen sections and paraffin sections (HE staining) and crystal formation. The pathological changes of the kidney. The kidney tissue was cut into 5 m ice sheet, detected by crystal components FTIR spectrometer.
3, the data were analyzed by SPSS 13 statistical software. The data were expressed as x + s. Urine data and blood data were analyzed by repeated measure variance analysis and factorial ANOVA. When P0.05, the difference was statistically significant.
Result锛，

本文编号：1374772