本文關(guān)鍵詞：三聚氰胺及三聚氰酸誘導(dǎo)大鼠腎臟損害及結(jié)石的研究　出處：《南方醫(yī)科大學(xué)》2010年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 三聚氰胺 三聚氰酸 三聚氰胺氰尿酸鹽 尿酸結(jié)石 急性腎功能衰竭

【摘要】： 研究背景 研究顯示2007年3月美國大量寵物中毒發(fā)生急性腎衰事件,是由于寵物食物中混有8.4%的三聚氰胺和5.3%三聚氰酸；而導(dǎo)致我國2008年9月因服用“問題奶粉”出現(xiàn)近30萬嬰幼兒發(fā)生泌尿系結(jié)石及泌尿系異常的原因則是“問題奶粉”中含有大量濃度不等的三聚氰胺。研究二者誘導(dǎo)腎臟損害及泌尿系結(jié)石的病理過程和發(fā)病機(jī)制,尋找預(yù)防和治療此類腎臟損害和結(jié)石的有效方法,是目前國內(nèi)外專家們急需積極研究的熱點(diǎn)方向。 三聚氰胺(MA,英文名Melamine),簡稱三胺,常用分子式為C3N6H6,是一種三嗪類含氮雜環(huán)有機(jī)化合物。外觀為粉末狀純白色單斜棱晶體,微溶于水,低毒,大鼠口服的半數(shù)致死量大于3克/公斤體重。主要用于合成三聚氰胺甲醛樹脂,以制作復(fù)合板、塑料、涂料、商用過濾器、膠水或粘合劑以及餐具和廚具。在三聚氰胺制造過程中產(chǎn)生的雜質(zhì)可能會形成氰尿酸、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謝為氨和二氧化碳,經(jīng)細(xì)菌代謝,也可能會形成其同系物。 三聚氰酸(CA,英文名Cyanuric acid等)；又名氰尿酸,異氰脲酸等,是由尿素聚合而得的白色結(jié)晶,分子式：C3H3N3O3,亦是一種重要的三嗪類含氮雜環(huán)有機(jī)化合物。微溶于水,基本無毒,大鼠LD50為7.7g/kg。用于有機(jī)合成氰尿酸一甲醛樹脂、涂料、粘合劑、農(nóng)藥除草劑、金屬氰化緩蝕劑、高分子材料改性劑和用于藥物鹵三羥嗪的生產(chǎn)等。 而最近的研究則發(fā)現(xiàn)被包括美國在內(nèi)的許多國家廣泛用來消毒食品生產(chǎn)設(shè)備、蔬菜和水果的三氯甲烷(Trichloromelamine),經(jīng)分解能生成三聚氰胺；而另外一種叫環(huán)丙氨嗪(cyromazine)的農(nóng)藥也可以殘留在蔬菜和水果的表面進(jìn)而分解成三聚氰胺。一種叫二氯異氰尿酸(dichloroisocyanurates)的消毒劑則被用來作為飲用水和游泳池水的消毒劑,而其可以代謝生成三聚氰酸。故三聚氰胺和三聚氰酸與人類生活已經(jīng)密切接觸。 既往研究顯示三聚氰胺和三聚氰酸在哺乳動物體內(nèi)屬于惰性代謝,即在體內(nèi)不會被轉(zhuǎn)化,亦不會蓄積體內(nèi),均以原型從尿液中排出。其中三聚氰胺在體內(nèi)的半衰期約為2.7h,三聚氰酸為2.5h,因此泌尿系統(tǒng)容易成為三聚氰胺與三聚氰酸損害的靶器官。國外的動物實(shí)驗(yàn)顯示長期單獨(dú)大劑量攝入三聚氰胺或三聚氰酸導(dǎo)致膀胱癌、膀胱結(jié)石、腎小管上皮增生、擴(kuò)張及腎纖維化等。而2008年Dobson等人的研究證實(shí)了三聚氰胺和三聚氰酸同時混合攝入導(dǎo)致大鼠急性腎衰竭而死亡,且病理解剖提示腎小管堵塞了大量結(jié)晶,同時發(fā)現(xiàn)其他器官未受明顯影響,并通過檢測發(fā)現(xiàn)腎組織中的結(jié)晶是三聚氰胺和三聚氰酸1：1結(jié)合的晶體。另外Dobson等人的研究還證明了單獨(dú)攝入三聚氰胺或三聚氰酸并未對大鼠腎臟造成損害,而且三聚氰胺或三聚氰酸在體內(nèi)所能達(dá)到的濃度限制內(nèi)是不會對腎臟細(xì)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無致腎損害及結(jié)石的數(shù)據(jù)。 由于三聚氰胺含氮量較高,因而被非法添加到寵物飼料、牛奶以及其它蛋白制品中以虛假提高蛋白含量。然而純度不高的三聚氰胺往往混雜有三聚氰酸,或者在食品加工過程中三聚氰胺可以少量轉(zhuǎn)化成三聚氰酸。我國“問題奶粉”事件后質(zhì)檢總局隨后在全國開展了嬰幼兒奶粉專項(xiàng)檢查,其中在某品牌奶粉中檢測出三聚氰胺含量高達(dá)2563mg/kg,但未報道檢測出三聚氰酸。此外,北京大學(xué)一附院的研究發(fā)現(xiàn)暴露在三聚氰胺的患兒泌尿系結(jié)石發(fā)病率比正常嬰幼兒高出7倍,北京兒童醫(yī)院分析出“問題奶粉”患兒結(jié)石成分為“三聚氰胺和尿酸”,所以目前國內(nèi)一直認(rèn)為是三聚氰胺與尿酸造成嬰幼兒發(fā)生腎結(jié)石。 急性腎衰(ARF)的發(fā)病機(jī)制主要分為腎前性、腎性和腎后性,而三聚氰胺和三聚氰酸被證明無直接腎毒性,其對腎臟損害的主要原因可能是腎后性梗阻引起的ARF。泌尿系結(jié)石的種類和發(fā)病機(jī)制十分復(fù)雜,而目前研究證明與三聚氰胺相關(guān)的結(jié)石很可能與尿酸一類結(jié)石相關(guān),這可能與三嗪類化合物影響體內(nèi)代謝,以及其分子結(jié)構(gòu)更易誘導(dǎo)尿酸的趨向附生而形成結(jié)石有關(guān),故需要研究三聚氰胺和三聚氰酸對尿酸排泄量、尿PH值、尿量等誘發(fā)尿酸結(jié)石的關(guān)鍵因素的影響。 “問題奶粉”對泌尿系損害機(jī)制尚未完全明確,基于受檢測的問題奶粉中含三聚氰胺最高劑量為2563mg/kg,再根據(jù)嬰兒最小平均體重(6kg)、平均每日攝入奶粉量(150g)及大鼠與人體藥理換算公式(約6.3：1),我們計算出復(fù)制大鼠模型每天應(yīng)攝入最大三聚氰胺劑量約為403.7 mg/kg。同時參考國外的動物實(shí)驗(yàn)劑量,設(shè)計了本實(shí)驗(yàn)給藥劑量進(jìn)行研究。本實(shí)驗(yàn)采用三聚氰胺和三聚氰酸單獨(dú)及聯(lián)合灌胃給藥方法,建立大鼠腎臟損害模型進(jìn)行研究,旨在探明三聚氰胺及其同系物三聚氰酸對腎臟損害和誘發(fā)結(jié)石的機(jī)制,為進(jìn)一步尋求防治方法提供實(shí)驗(yàn)依據(jù)。 研究目的： 1、探討單獨(dú)的三聚氰胺或三聚氰酸對大鼠腎臟損害及誘發(fā)泌尿系結(jié)石的機(jī)制,并分析結(jié)石晶體成分。 2、探討三聚氰胺和三聚氰酸的聯(lián)合效應(yīng)損害大鼠腎臟及誘發(fā)泌尿系結(jié)石的機(jī)制,并分析結(jié)石晶體成分。 3、通過大鼠的實(shí)驗(yàn)研究來探討“問題奶粉”致嬰幼兒泌尿系結(jié)石的可能機(jī)制。 研究方法： 1、制作模型和分組 將60只SD大鼠隨機(jī)分成A組(空白對照組)、B組(三聚氰胺400mg·kg-1·d-1)、C組(三聚氰酸400mg·kg-1·d-1)、D組(三聚氰胺400mg·kg-1·d-1 +三聚氰酸400mg·kg-1·d-1)、E組(三聚氰胺30mg·kg-1·d-1+三聚氰酸30mg·kg-1·d-1),每組12只,通過灌胃給藥。 2、指標(biāo)的收集和檢測 用代謝籠收集前3天24小時尿液,進(jìn)行24小時尿量、PH值和24小時尿酸排泄量測量及尿沉渣涂片檢查；分別于第4天、第31天灌胃前每組采集6只大鼠的血液標(biāo)本檢測血肌酐、尿素氮、尿酸；取雙側(cè)腎臟用外科顯微鏡觀察后,制作快速冰凍切片及石蠟切片(HE染色)觀察腎臟病理改變及晶體形成。腎臟冰凍組織切成5μm薄片后,用紅外顯微光譜儀檢測晶體成分。 3、采用SPSS 13.0統(tǒng)計軟件進(jìn)行數(shù)據(jù)分析,數(shù)據(jù)以x±s表示。尿液數(shù)據(jù)和血液數(shù)據(jù)分別采用重復(fù)測量方差分析和析因方差分析。當(dāng)P0.05時,差異有統(tǒng)計學(xué)意義。 結(jié)果： 1、尿液和血液生化結(jié)果 與A組比較,B組和C組大鼠的24h尿尿量、24h尿酸排泄量均明顯增多,差異均有統(tǒng)計學(xué)意義(P0.05)；而D、E組尿量明顯減少、PH值降低、24h尿酸排泄量明顯減少,差異均有統(tǒng)計學(xué)意義(P0.05),其中D組變化比E組更為明顯,差異有統(tǒng)計學(xué)意義(P0.05)。在相應(yīng)時間點(diǎn)各組與A組比較,B組和C組的血肌酐、血尿酸及血尿素氮均升高,但在第4天時候僅血尿素氮差異有統(tǒng)計學(xué)意義(P0.05),而第31天血尿素氮及血尿酸差異均有統(tǒng)計學(xué)意義(P0.05)；D組在第4天的時候各項(xiàng)指標(biāo)均顯著升高,差異有統(tǒng)計學(xué)意義(P0.05)；E組第4天血肌酐及血尿素氮明顯升高,差異有統(tǒng)計學(xué)意義(P0.05),第31天則各項(xiàng)指標(biāo)明顯升高,差異均有統(tǒng)計學(xué)意義(P0.05)。其中D組比E組變化更為明顯,差異有統(tǒng)計學(xué)意義(P0.05),同時E組各項(xiàng)指標(biāo)第31天比第4天顯著升高,差異有統(tǒng)計學(xué)意義(P0.05)。(見表2) 2、尿沉渣涂片 從尿沉渣涂片中可見：A組尿液無明顯晶體；B組可見疑似三聚氰胺原型的晶體；C組無明顯晶體,但有少許細(xì)沙樣小顆粒沉淀物；D組及E組均可見淡黃色圓形特異性晶體。 3、腎臟病理改變 大體標(biāo)本：A組大鼠腎臟包膜光滑暗紅色,腎臟解剖觀察無明顯病理改變。B、C組大鼠腎臟皮髓結(jié)構(gòu)清晰,腎盞及腎盂良好,未見結(jié)石晶體沉積。D組大鼠腎臟明顯腫大,外觀呈灰黃色,包膜有許多散在黃色斑點(diǎn),腎臟皮質(zhì)變薄,腎盞、腎盂結(jié)構(gòu)破壞,腎內(nèi)布滿淡黃色結(jié)石晶體。E組第4天皮髓結(jié)構(gòu)尚清晰,可見不少淡黃色結(jié)石晶體沉積；第31天E組大鼠腎臟剖面呈紅褐色,皮髓變模糊,大量淡黃色顆粒沉積。各組大鼠膀胱均未見觀察到結(jié)石。 腎組織切片：A組大鼠未見明顯病理改變。B組及C組腎組織石蠟切片與冰凍切片在第4天的時候均未見結(jié)晶及明顯病理改變；第31天亦未見晶體,僅見少量腎小管輕度擴(kuò)張、上皮細(xì)胞腫脹和少量管型形成等。D組與E組腎組織石蠟切片與冰凍切片均可見淡黃色、呈放射狀的同心圓形晶體,主要集聚于腎小管,也見于集合管、間質(zhì)及被膜下,偶見于腎小球囊中,大多晶體以不規(guī)則碎片出現(xiàn)于腎小管中。此外,腎小管濁腫、上皮受損,并有各種管型形成,大量炎性細(xì)胞在腎小球囊腔、腎小管及間質(zhì)中；E組第31天還可看到許多纖維細(xì)胞充滿間質(zhì)。此外第4’天D組切片單倍視野下結(jié)石晶體數(shù)量和體積,以及組織病理變化都大于E組；而第31天E組結(jié)石晶體數(shù)量、體積以及組織病理變化都比其在第4天觀察到的要大。 4、腎組織晶體成分分析結(jié)果 對混合組大鼠腎臟出現(xiàn)的各種形態(tài)結(jié)石晶體進(jìn)行紅外顯微光譜分析,得到基本一致的紅外光譜圖；根據(jù)專業(yè)分析,及查閱相關(guān)文獻(xiàn)可知混合藥物組大鼠在腎臟生成的晶體即為三聚氰胺氰尿酸鹽晶體。 結(jié)論： 1、三聚氰胺和三聚氰酸同時混合進(jìn)入體內(nèi),會在腎臟形成一種不溶于水的三聚氰胺氰尿酸鹽晶體,此晶體能堵塞腎小管造成腎臟損害甚至急性腎衰竭,并可以作為結(jié)石核心與尿酸等成分一起誘發(fā)泌尿系結(jié)石； 2、三聚氰胺與其同系物三聚氰酸對腎臟的損害較輕,機(jī)制相似；單獨(dú)攝入三聚氰胺或三聚氰酸,大鼠腎臟無結(jié)石晶體形成,但長期攝入會對腎功能造成損害,其原因?yàn)殚L期的滲透性利尿?qū)е挛锢硇該p傷腎組織； 3、三聚氰胺與三聚氰酸的聯(lián)合效應(yīng)比它們的單獨(dú)效應(yīng)對腎臟的損害作用更大,更容易誘發(fā)泌尿系尿酸結(jié)石；其聯(lián)合效應(yīng)與聯(lián)合藥物的濃度和暴露時間成正相關(guān)。 4、“問題奶粉”致嬰幼兒泌尿系結(jié)石的機(jī)制較復(fù)雜,但奶粉中同時存在三聚氰胺和三聚氰酸可能是主要誘發(fā)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錛，

本文編號：1374772