三聚氰胺与三聚氰酸的联合毒性作用研究
发布时间：2018-11-06 14:21
【摘要】：2007年春天,美国爆发大规模宠物因食用受三聚氰胺污染的饲料而发生急性肾衰竭事件。截至到4月,美国食品药物管理局(FDA)已收到至少471例动物发生肾衰竭的报告,其中约有104只猫和狗因食用受污染的食物而出现死亡现象。无独有偶,2008年9月中国报道了婴幼儿食用蓄意添加三聚氰胺的奶粉出现肾结石。根据我国官方公布的数字,截至2008年9月21日,因使用含三聚氰胺奶粉而患病的婴幼儿累计达294,000人,其中有51,000名儿童急需住院治疗,此前已治愈出院1,579人,死亡6人,给受害者家庭造成了沉重的打击。事件引起各国的高度关注和对乳制品安全的担忧,中国乳制品行业也遭到了重创。三聚氰胺及其同系物的毒性备受关注。 本文采用体内与体外实验相结合的方法研究三聚氰胺与及其同系物三聚氰酸的联合毒性。体内试验包括三聚氰胺与三聚氰酸对小鼠的联合急性毒性和骨髓微核实验,体外试验研究了三聚氰胺与三聚氰酸对人体肝细胞株(L02)和肾细胞株(293)的活性和对肾损伤信号因子kim-1表达水平的影响。 采用寇氏法研究三聚氰胺与三聚氰酸(M+C)对小鼠的联合急性毒性作用,M+C按1：1、2：1和1：2三种比例混合经口一次性灌胃小鼠。每种比例设6个剂量组即109、173、274、435、689和1092mg/kg,灌胃容量为0.02mL/g。并设溶剂对照组和空白组。结果：M+C(1：1)组,雄鼠LD50为274mg/kg,雌鼠LD50为401mg/kg;M+C(2:1)组,雄鼠LD50值为401mg/kg、雌鼠LD50值为546mg/kg;M+C(1:2)组,雄鼠LD50值为344mg/kg、雌鼠LD50值为589mg/kg。M+C三种比例混合物经口灌胃,剂量为1092mg/kg时,动物均死亡；剂量低于173mg/kg时,各组动物均无死亡。M+C各给药组,小鼠肾脏均发生病变,肾脏不同程度膨胀,表面可见斑点,肾脏脏器系数值大于两个对照组,差异显著(P0.05)。 根据联合急性毒性试验结果,设置受试物的三个剂量组：高剂量组(1/2LD50)中剂量组(1/4LD50)、低剂量组(1/8LD50),采用间隔24h灌胃染毒法,探讨三种质量比下的三聚氰胺与三聚氰酸混合物对细胞染色体损伤的影响。微核试验结果表明：不同质量比的三聚氰胺与三聚氰酸混合物各剂量组微核数与阴性对照组相比均无显著性增加(P0.05),受试物对小鼠骨髓微核试验结果为阴性。 采用MTT法研究三聚氰胺与三聚氰酸混合物对肝细胞株(L02)和肾细胞株(293)的细胞活性影响,三聚氰胺与三聚氰酸按1：1、2：1和1：2三种质量比比例混合,浓度分别为0、5.1、10.3、20.6、41.2、82.5和165μg/ml。体外细胞毒性实验结果表明：M+C(1:1)、M+C (2:1)和M+C(1：2)组在剂量为165μg/ml时对L02肝细胞和293肾细胞活性都有抑制作用。随着浓度的降低三种比例下的混合物对L02肝细胞和293肾细胞活性影响逐渐减小。低剂量2.156μg/ml组对L02肝细胞和293肾细胞活性有一定的促进作用。 蛋白免疫印迹法(Western blot)研究三聚氰胺与三聚氰酸混合物对293肾细胞损伤信号因子kim-1表达水平的影响。三聚氰胺与三聚氰酸采用1：1质量比例,在MTT的试验基础上,选择了三种剂量组82.5、41.25和20.625gg/ml,另设空白组。结果表明：给予三聚氰胺与三聚氰酸混合物(1：1)剂量为82.5μg/ml和41.25μg/m1l时,细胞Kim-1蛋白表达明显升高(P0.05)。而20.625μg/ml剂量组的Kim-1蛋白表达量与对照组相比无显著性差异(P0.05)。
[Abstract]:In the spring of 2007, a large-scale pet in the United States had an acute renal failure event due to the consumption of melamine-contaminated feed. As of April, the Food and Drug Administration of the United States (FDA) had received at least 471 reports of renal failure in animals, of which about 104 cats and dogs died of contaminated food. No one, in September 2008, China reported that infant consumption of melamine-added milk powder had a kidney stone. According to the figures released by our authorities, as of 21 September 2008, a total of 294,000 children with the use of melamine-containing milk powder had a total of 294,000 children, of which 51,000 children were in urgent need of hospitalization, which had previously been cured with 1,579 people and six deaths, resulting in a heavy blow to the families of the victims. China's dairy industry has also been hit hard by the high concerns of countries and concerns about the safety of dairy products. The toxicity of melamine and its homologs is of great concern. In this paper, a combination of in vivo and in vitro experiments was used to study the combination of melamine with its homolog cyanuric acid. The effect of melamine and cyanuric acid on the human liver cell line (L02) and the renal cell line (293) and the expression level of the kidney injury signal factor kim-1 were studied in vitro. The effects of melamine and cyanuric acid (M + C) on the combined acute toxicity of the mice were studied by means of the method of the method of Kohlet. M + C was mixed in 1: 1, 2: 1 and 1: 2 ratio at 1: 1, 2: 1 and 1: 2. In each proportion, 6 dose groups, 109, 173, 274, 435, 689 and 1092mg/ kg, were set in each proportion, and the gastric administration capacity was 0. 0. 2mL/ g. and set with a solvent control Results: The LD50 of the M + C (1: 1) group was 274mg/ kg, the LD50 of the female was 401mg/ kg, the LD50 value of the female was 401mg/ kg, the LD50 value of the female was 546mg/ kg, the LD50 value of the M + C (1: 2) group was 344mg/ kg, the LD50 value of the female was 589mg/ kg. The animals were all dead; when the dose was lower than 173mg/ kg, each group There was no death in both of M + C and the kidney of the mice. The kidney of the mice was expanded, the surface was visible, and the number of the organs of the kidney was greater than that of the two control groups, and the difference was significant (P 0.05). Based on the results of the combined acute toxicity test, the three dose groups of the subject were set: the dose group (1/ 4LD50) in the high dose group (1/ 2LD50), the low dose group (1/ 8LD50), and the interval 24. The mixture of melamine and cyanuric acid in three mass ratios was studied by the method of intragastric administration. The results of micronucleus test showed that there was no significant increase in the number of micronuclei in each dose group of melamine and cyanuric acid mixture in different mass ratios (P0.05). The effects of the mixture of melamine and cyanuric acid on the cell activity of the liver cell line (L02) and the renal cell line (293) were studied by MTT method. The ratio of melamine to cyanuric acid was 1: 1, 2: 1 and 1: 2. The concentrations were 0, 5. 1, 10. 3, 20. 6, 41. 2, 82, respectively. The results of in vitro cytotoxicity test showed that M + C (1: 1), M + C (2: 1) and M + C (1: 2) group had a dose of 165. m u.g/ ml to L02 hepatocytes and 293 cells. There was an inhibitory effect on the activity of the renal cell. The mixture of the three proportions with the decrease of the concentration on the L02 hepatocytes and 293 The effect of the activity of the renal cell was gradually decreased. The low dose of 2. 156. m u.g/ ml was used to fine the L02 hepatocytes and the 293 cells. The effect of cyanuric acid and the mixture of melamine and cyanuric acid on the cell injury of 293 cells was studied by Western blot. The effect of the expression level of factor kim-1. The ratio of melamine to cyanuric acid was 1: 1, and three dose groups of 82.5, 41.25 and 20 were selected on the basis of the MTT assay. The results showed that when the dosage of melamine and cyanuric acid mixture (1: 1) was 82.5 & mu; g/ ml and 40.25 & mu; g/ m1l, the cell Kim-1 egg The expression of the Kim-1 protein in the 20. 625. mu.g/ ml dose group was significantly higher than that in the control group (P0.05).
【学位授予单位】：武汉轻工大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：TS201.6;R114
本文编号：2314537
[Abstract]:In the spring of 2007, a large-scale pet in the United States had an acute renal failure event due to the consumption of melamine-contaminated feed. As of April, the Food and Drug Administration of the United States (FDA) had received at least 471 reports of renal failure in animals, of which about 104 cats and dogs died of contaminated food. No one, in September 2008, China reported that infant consumption of melamine-added milk powder had a kidney stone. According to the figures released by our authorities, as of 21 September 2008, a total of 294,000 children with the use of melamine-containing milk powder had a total of 294,000 children, of which 51,000 children were in urgent need of hospitalization, which had previously been cured with 1,579 people and six deaths, resulting in a heavy blow to the families of the victims. China's dairy industry has also been hit hard by the high concerns of countries and concerns about the safety of dairy products. The toxicity of melamine and its homologs is of great concern. In this paper, a combination of in vivo and in vitro experiments was used to study the combination of melamine with its homolog cyanuric acid. The effect of melamine and cyanuric acid on the human liver cell line (L02) and the renal cell line (293) and the expression level of the kidney injury signal factor kim-1 were studied in vitro. The effects of melamine and cyanuric acid (M + C) on the combined acute toxicity of the mice were studied by means of the method of the method of Kohlet. M + C was mixed in 1: 1, 2: 1 and 1: 2 ratio at 1: 1, 2: 1 and 1: 2. In each proportion, 6 dose groups, 109, 173, 274, 435, 689 and 1092mg/ kg, were set in each proportion, and the gastric administration capacity was 0. 0. 2mL/ g. and set with a solvent control Results: The LD50 of the M + C (1: 1) group was 274mg/ kg, the LD50 of the female was 401mg/ kg, the LD50 value of the female was 401mg/ kg, the LD50 value of the female was 546mg/ kg, the LD50 value of the M + C (1: 2) group was 344mg/ kg, the LD50 value of the female was 589mg/ kg. The animals were all dead; when the dose was lower than 173mg/ kg, each group There was no death in both of M + C and the kidney of the mice. The kidney of the mice was expanded, the surface was visible, and the number of the organs of the kidney was greater than that of the two control groups, and the difference was significant (P 0.05). Based on the results of the combined acute toxicity test, the three dose groups of the subject were set: the dose group (1/ 4LD50) in the high dose group (1/ 2LD50), the low dose group (1/ 8LD50), and the interval 24. The mixture of melamine and cyanuric acid in three mass ratios was studied by the method of intragastric administration. The results of micronucleus test showed that there was no significant increase in the number of micronuclei in each dose group of melamine and cyanuric acid mixture in different mass ratios (P0.05). The effects of the mixture of melamine and cyanuric acid on the cell activity of the liver cell line (L02) and the renal cell line (293) were studied by MTT method. The ratio of melamine to cyanuric acid was 1: 1, 2: 1 and 1: 2. The concentrations were 0, 5. 1, 10. 3, 20. 6, 41. 2, 82, respectively. The results of in vitro cytotoxicity test showed that M + C (1: 1), M + C (2: 1) and M + C (1: 2) group had a dose of 165. m u.g/ ml to L02 hepatocytes and 293 cells. There was an inhibitory effect on the activity of the renal cell. The mixture of the three proportions with the decrease of the concentration on the L02 hepatocytes and 293 The effect of the activity of the renal cell was gradually decreased. The low dose of 2. 156. m u.g/ ml was used to fine the L02 hepatocytes and the 293 cells. The effect of cyanuric acid and the mixture of melamine and cyanuric acid on the cell injury of 293 cells was studied by Western blot. The effect of the expression level of factor kim-1. The ratio of melamine to cyanuric acid was 1: 1, and three dose groups of 82.5, 41.25 and 20 were selected on the basis of the MTT assay. The results showed that when the dosage of melamine and cyanuric acid mixture (1: 1) was 82.5 & mu; g/ ml and 40.25 & mu; g/ m1l, the cell Kim-1 egg The expression of the Kim-1 protein in the 20. 625. mu.g/ ml dose group was significantly higher than that in the control group (P0.05).
【学位授予单位】：武汉轻工大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：TS201.6;R114
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