三聚氰胺与三聚氰酸联用对鸡肾脏损伤的研究
发布时间：2018-11-11 00:48
【摘要】：三聚氰胺与三聚氰酸是两种化工原料,均不能添加到饲料中。但近年来由于工业污染、饲料兽药原料生产、环境富集等因素,三聚氰胺与三聚氰酸常被发现存在于饲料中,给动物带来严重的安全隐患。为进一步明确三聚氰胺与三聚氰酸联用对鸡肾脏的损伤提供试验依据,本试验将144只7日龄蛋雏鸡随机均分成三组,对照组饲喂基础饲料,低剂量组饲喂[(30 mg三聚氰胺+10 mg三聚氰酸)/kg]的饲料、高剂量组饲喂[(50 mg三聚氰胺+16.7 mg三聚氰酸)/kg]的饲料。在试验开始后六周内,每周颈部脱臼处死对照组、实验组鸡群(8只/组/次),取出肾脏,通过亚硝酸盐法测定T-SOD活性、硫代巴比妥酸反应法测定MDA含量、常规石蜡切片HE染色方法、细胞凋亡染液Hoechst 33258切片染色方法、荧光分光光度计分析法定量检测Caspase-3活性、免疫组化法检测GRP78蛋白的表达来研究三聚氰胺与三聚氰酸联用对鸡肾脏的损伤。研究结果如下:1.所有实验组鸡群肾脏T-SOD活性均值低于对照组。其中试验第1周至第5周,实验组与对照组相比差异不显著(P0.05),而在试验第6周,高剂量实验组鸡群肾脏T-SOD活性均值显著低于于对照组(P0.05);所有实验组MDA含量均高于对照组,其中第3周至第6周实验组(第6周低剂量组除外)MDA含量均值均显著高于对照组(P0.01)。结果表明二者联用能诱导鸡肾脏氧化损伤,与时间和剂量有一定关系。2.低剂量组:第1周与第3周肾小管上皮细胞出现轻微病变;第6周可见肾小管上皮细胞肿胀、变性,一些细胞细胞核浓缩。高剂量组:第1周病变较轻微,第3周肾小管上皮细胞肿胀、坏死。第6周肾小管上皮细胞细胞胞核崩解、淡染、消失;细胞核浓缩;上皮细胞与基底膜分离,细胞脱落到管腔内;肾间质毛细血管扩张并伴有轻度出血。结果表明二者联用能导致鸡肾脏组织病理损伤,主要集中在对肾小管的损伤,可引起细胞的变性、坏死和细胞凋亡。3.试验第6周实验组凋亡细胞数与对照组相比显著增多;实验组鸡群肾脏组织内Caspase-3活性显著高于对照组(P0.05)。结果表明二者联用能诱导鸡肾脏组织内Caspase-3活性增高,进而引起凋亡细胞的数目增多。4.试验第6周三聚氰胺与三聚氰酸联用的实验组GRP78蛋白表达量与对照相比显著增高(P0.01)。结果表明二者联用能诱导鸡肾脏细胞GRP78蛋白的表达量显著增加,揭示存在内质网应激反应。
[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.
【学位授予单位】：河南科技大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S859.8
本文编号：2324038
[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.
【学位授予单位】：河南科技大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S859.8
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