三聚氰胺與三聚氰酸的聯(lián)合毒性作用研究
發(fā)布時(shí)間：2018-11-06 14:21
【摘要】：2007年春天,美國爆發(fā)大規(guī)模寵物因食用受三聚氰胺污染的飼料而發(fā)生急性腎衰竭事件。截至到4月,美國食品藥物管理局(FDA)已收到至少471例動(dòng)物發(fā)生腎衰竭的報(bào)告,其中約有104只貓和狗因食用受污染的食物而出現(xiàn)死亡現(xiàn)象。無獨(dú)有偶,2008年9月中國報(bào)道了嬰幼兒食用蓄意添加三聚氰胺的奶粉出現(xiàn)腎結(jié)石。根據(jù)我國官方公布的數(shù)字,截至2008年9月21日,因使用含三聚氰胺奶粉而患病的嬰幼兒累計(jì)達(dá)294,000人,其中有51,000名兒童急需住院治療,此前已治愈出院1,579人,死亡6人,給受害者家庭造成了沉重的打擊。事件引起各國的高度關(guān)注和對(duì)乳制品安全的擔(dān)憂,中國乳制品行業(yè)也遭到了重創(chuàng)。三聚氰胺及其同系物的毒性備受關(guān)注。 本文采用體內(nèi)與體外實(shí)驗(yàn)相結(jié)合的方法研究三聚氰胺與及其同系物三聚氰酸的聯(lián)合毒性。體內(nèi)試驗(yàn)包括三聚氰胺與三聚氰酸對(duì)小鼠的聯(lián)合急性毒性和骨髓微核實(shí)驗(yàn),體外試驗(yàn)研究了三聚氰胺與三聚氰酸對(duì)人體肝細(xì)胞株(L02)和腎細(xì)胞株(293)的活性和對(duì)腎損傷信號(hào)因子kim-1表達(dá)水平的影響。 采用寇氏法研究三聚氰胺與三聚氰酸(M+C)對(duì)小鼠的聯(lián)合急性毒性作用,M+C按1：1、2：1和1：2三種比例混合經(jīng)口一次性灌胃小鼠。每種比例設(shè)6個(gè)劑量組即109、173、274、435、689和1092mg/kg,灌胃容量為0.02mL/g。并設(shè)溶劑對(duì)照組和空白組。結(jié)果：M+C(1：1)組,雄鼠LD50為274mg/kg,雌鼠LD50為401mg/kg;M+C(2:1)組,雄鼠LD50值為401mg/kg、雌鼠LD50值為546mg/kg;M+C(1:2)組,雄鼠LD50值為344mg/kg、雌鼠LD50值為589mg/kg。M+C三種比例混合物經(jīng)口灌胃,劑量為1092mg/kg時(shí),動(dòng)物均死亡；劑量低于173mg/kg時(shí),各組動(dòng)物均無死亡。M+C各給藥組,小鼠腎臟均發(fā)生病變,腎臟不同程度膨脹,表面可見斑點(diǎn),腎臟臟器系數(shù)值大于兩個(gè)對(duì)照組,差異顯著(P0.05)。 根據(jù)聯(lián)合急性毒性試驗(yàn)結(jié)果,設(shè)置受試物的三個(gè)劑量組：高劑量組(1/2LD50)中劑量組(1/4LD50)、低劑量組(1/8LD50),采用間隔24h灌胃染毒法,探討三種質(zhì)量比下的三聚氰胺與三聚氰酸混合物對(duì)細(xì)胞染色體損傷的影響。微核試驗(yàn)結(jié)果表明：不同質(zhì)量比的三聚氰胺與三聚氰酸混合物各劑量組微核數(shù)與陰性對(duì)照組相比均無顯著性增加(P0.05),受試物對(duì)小鼠骨髓微核試驗(yàn)結(jié)果為陰性。 采用MTT法研究三聚氰胺與三聚氰酸混合物對(duì)肝細(xì)胞株(L02)和腎細(xì)胞株(293)的細(xì)胞活性影響,三聚氰胺與三聚氰酸按1：1、2：1和1：2三種質(zhì)量比比例混合,濃度分別為0、5.1、10.3、20.6、41.2、82.5和165μg/ml。體外細(xì)胞毒性實(shí)驗(yàn)結(jié)果表明：M+C(1:1)、M+C (2:1)和M+C(1：2)組在劑量為165μg/ml時(shí)對(duì)L02肝細(xì)胞和293腎細(xì)胞活性都有抑制作用。隨著濃度的降低三種比例下的混合物對(duì)L02肝細(xì)胞和293腎細(xì)胞活性影響逐漸減小。低劑量2.156μg/ml組對(duì)L02肝細(xì)胞和293腎細(xì)胞活性有一定的促進(jìn)作用。 蛋白免疫印跡法(Western blot)研究三聚氰胺與三聚氰酸混合物對(duì)293腎細(xì)胞損傷信號(hào)因子kim-1表達(dá)水平的影響。三聚氰胺與三聚氰酸采用1：1質(zhì)量比例,在MTT的試驗(yàn)基礎(chǔ)上,選擇了三種劑量組82.5、41.25和20.625gg/ml,另設(shè)空白組。結(jié)果表明：給予三聚氰胺與三聚氰酸混合物(1：1)劑量為82.5μg/ml和41.25μg/m1l時(shí),細(xì)胞Kim-1蛋白表達(dá)明顯升高(P0.05)。而20.625μg/ml劑量組的Kim-1蛋白表達(dá)量與對(duì)照組相比無顯著性差異(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).
【學(xué)位授予單位】：武漢輕工大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類號(hào)】：TS201.6;R114
本文編號(hào)：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).
【學(xué)位授予單位】：武漢輕工大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類號(hào)】：TS201.6;R114
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