【摘要】：三聚氰胺與三聚氰酸是兩種化工原料,均不能添加到飼料中。但近年來由于工業(yè)污染、飼料獸藥原料生產(chǎn)、環(huán)境富集等因素,三聚氰胺與三聚氰酸常被發(fā)現(xiàn)存在于飼料中,給動(dòng)物帶來嚴(yán)重的安全隱患。為進(jìn)一步明確三聚氰胺與三聚氰酸聯(lián)用對(duì)雞腎臟的損傷提供試驗(yàn)依據(jù),本試驗(yàn)將144只7日齡蛋雛雞隨機(jī)均分成三組,對(duì)照組飼喂基礎(chǔ)飼料,低劑量組飼喂[(30 mg三聚氰胺+10 mg三聚氰酸)/kg]的飼料、高劑量組飼喂[(50 mg三聚氰胺+16.7 mg三聚氰酸)/kg]的飼料。在試驗(yàn)開始后六周內(nèi),每周頸部脫臼處死對(duì)照組、實(shí)驗(yàn)組雞群(8只/組/次),取出腎臟,通過亞硝酸鹽法測(cè)定T-SOD活性、硫代巴比妥酸反應(yīng)法測(cè)定MDA含量、常規(guī)石蠟切片HE染色方法、細(xì)胞凋亡染液Hoechst 33258切片染色方法、熒光分光光度計(jì)分析法定量檢測(cè)Caspase-3活性、免疫組化法檢測(cè)GRP78蛋白的表達(dá)來研究三聚氰胺與三聚氰酸聯(lián)用對(duì)雞腎臟的損傷。研究結(jié)果如下:1.所有實(shí)驗(yàn)組雞群腎臟T-SOD活性均值低于對(duì)照組。其中試驗(yàn)第1周至第5周,實(shí)驗(yàn)組與對(duì)照組相比差異不顯著(P0.05),而在試驗(yàn)第6周,高劑量實(shí)驗(yàn)組雞群腎臟T-SOD活性均值顯著低于于對(duì)照組(P0.05);所有實(shí)驗(yàn)組MDA含量均高于對(duì)照組,其中第3周至第6周實(shí)驗(yàn)組(第6周低劑量組除外)MDA含量均值均顯著高于對(duì)照組(P0.01)。結(jié)果表明二者聯(lián)用能誘導(dǎo)雞腎臟氧化損傷,與時(shí)間和劑量有一定關(guān)系。2.低劑量組:第1周與第3周腎小管上皮細(xì)胞出現(xiàn)輕微病變;第6周可見腎小管上皮細(xì)胞腫脹、變性,一些細(xì)胞細(xì)胞核濃縮。高劑量組:第1周病變較輕微,第3周腎小管上皮細(xì)胞腫脹、壞死。第6周腎小管上皮細(xì)胞細(xì)胞胞核崩解、淡染、消失;細(xì)胞核濃縮;上皮細(xì)胞與基底膜分離,細(xì)胞脫落到管腔內(nèi);腎間質(zhì)毛細(xì)血管擴(kuò)張并伴有輕度出血。結(jié)果表明二者聯(lián)用能導(dǎo)致雞腎臟組織病理?yè)p傷,主要集中在對(duì)腎小管的損傷,可引起細(xì)胞的變性、壞死和細(xì)胞凋亡。3.試驗(yàn)第6周實(shí)驗(yàn)組凋亡細(xì)胞數(shù)與對(duì)照組相比顯著增多;實(shí)驗(yàn)組雞群腎臟組織內(nèi)Caspase-3活性顯著高于對(duì)照組(P0.05)。結(jié)果表明二者聯(lián)用能誘導(dǎo)雞腎臟組織內(nèi)Caspase-3活性增高,進(jìn)而引起凋亡細(xì)胞的數(shù)目增多。4.試驗(yàn)第6周三聚氰胺與三聚氰酸聯(lián)用的實(shí)驗(yàn)組GRP78蛋白表達(dá)量與對(duì)照相比顯著增高(P0.01)。結(jié)果表明二者聯(lián)用能誘導(dǎo)雞腎臟細(xì)胞GRP78蛋白的表達(dá)量顯著增加,揭示存在內(nèi)質(zhì)網(wǎng)應(yīng)激反應(yīng)。
[Abstract]:Melamine and cyanuric acid are two kinds of chemical raw materials and can not be added to feed. However, in recent years, due to industrial pollution, raw material production of feed veterinary drugs, environmental enrichment and other factors, melamine and cyanuric acid are often found in the feed, which brings serious safety risks to animals. In order to further clarify the experimental basis of kidney injury induced by melamine and cyanuric acid, 144 7-day-old egg chicks were randomly divided into three groups, the control group was fed with basic feed. The low dose group was fed with [(30 mg melamine 10 mg cyanuric acid) / kg] and the high dose group [(50 mg melamine 16.7 mg cyanuric acid) / kg]. Within six weeks after the trial, the control group was killed by cervical dislocations, and the chickens of the experimental group (8 chickens / group) were removed. The T-SOD activity was measured by nitrite method, and the content of MDA was measured by thiobarbituric acid reaction. The HE staining method of paraffin section, the Hoechst 33258 section staining method of cell apoptosis, and the quantitative detection of Caspase-3 activity by fluorescence spectrophotometer were used. The expression of GRP78 protein was detected by immunohistochemical method to study the kidney damage induced by melamine combined with cyanuric acid. The results are as follows: 1. The mean value of renal T-SOD activity in all experimental groups was lower than that in control group. From week 1 to week 5, there was no significant difference between the experimental group and the control group (P0.05), but at the 6th week, the average of renal T-SOD activity in the high-dose experimental group was significantly lower than that in the control group (P0.05). The MDA content in all experimental groups was higher than that in control group, and the mean MDA content in experimental group (except low-dose group at 6th week) was significantly higher than that in control group (P0.01) from the 3rd week to the 6th week. The results showed that combined treatment could induce oxidative damage of chicken kidney, which was related to time and dose. 2. In the low dose group, the renal tubular epithelial cells showed slight pathological changes at week 1 and week 3, and tubule epithelial cells were swollen and denatured at the 6th week, and some nuclei were concentrated. In high dose group, the lesion was mild at week 1 and tubule epithelial cell swelling and necrosis at week 3. At the 6th week, the nucleus of renal tubular epithelial cells disintegrated, stained and disappeared; the nucleus was concentrated; the epithelial cells separated from the basement membrane and the cells fell off into the lumen; the renal interstitial capillaries were dilated with mild hemorrhage. The results showed that the combination of the two methods could cause pathological damage of chicken kidney, mainly focusing on the injury of renal tubules, which could cause degeneration, necrosis and apoptosis of cells. At the 6th week, the number of apoptotic cells in the experimental group was significantly higher than that in the control group, and the activity of Caspase-3 in the kidney tissue of the experimental group was significantly higher than that of the control group (P0.05). The results showed that the combination of the two could induce the increase of Caspase-3 activity in the kidney tissue, and then the number of apoptotic cells increased. 4. The expression of GRP78 protein in the experimental group treated with melamine and cyanuric acid was significantly higher than that in the control group at the 6th week (P0.01). The results showed that the expression of GRP78 protein was significantly increased in chicken kidney cells induced by the combination of the two methods, which revealed the presence of endoplasmic reticulum stress response.
【學(xué)位授予單位】：河南科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S859.8

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