三聚氰胺及三聚氰酸混合物对小鼠肝脏损伤的研究
摘要
研究目的：本实验为了探讨三聚氰胺及三聚氰酸混合物对小鼠肝脏组织的毒性损伤作用,并查明其作用机理,从而完善其毒性研究,并全面地客观地评价三聚氰胺及三聚氰酸的毒性作用,为动物饲料生产安全提供理论参考依据。
研究方法：本实验采用25-30日龄小白鼠为试验对象,通过三聚氰胺及三聚氰酸灌胃攻毒30天后,断颈椎处死,眼眶采血并取出肝脏进行如下实验：采用常规方法检测三聚氰胺及三聚氰酸对肝功能的影响；检测肝脏组织中超氧化物歧化酶(SOD)的活性及丙二醛(MDA)的含量；采用制作病理组织切片法检查小鼠肝组织病理变化；采用RT-PCR法检测凋亡相关调控基因Bax、Bcl-2、Caspase-3的表达；采用TUNEL法检测肝脏细胞是否凋亡及凋亡率的变化,并采用免疫组织化学法检测三聚氰胺及三聚氰酸对凋亡相关调控基因Bax、Bcl-2、Caspase-3蛋白表达的影响。
研究结果：攻毒后,三聚氰胺和三聚氰酸混合各剂量组小鼠均出现不同程度的肝细胞变性、坏死,细胞核浓缩、碎裂、消失,有些严重甚至出现坏死灶。随着攻毒剂量的增加MDA的含量逐渐升高,而SOD活性降低。肝功能检测中,各剂量组ALT、AST活力随着三聚氰胺和三聚氰酸混合物剂量的增加而逐渐升高,TP含量随之而降低。TUNEL法检测结果显示：各混合剂量组均有不同程度的凋亡,且随着浓度升高,细胞凋亡越明显。RT-PCR结果显示Bax和caspase-3基因表达水平明显上升,Bcl-2基因表达水平明显下降,混合高剂量组与对照组比较OD值均明显降低差异极显著(P<0.01)。免疫组化结果显示：Bax和caspase-3都参与了三聚氰胺和三聚氰酸诱导小鼠肝细胞凋亡的调控,而Bcl-2与三聚氰胺和三聚氰酸诱导小鼠肝细胞凋亡无关。
研究结论：三聚氰胺和三聚氰酸混合物对小鼠肝脏有损伤作用,随着混合物剂量的上升呈一定的量效关系,剂量越高损伤情况越严重。而单独的三聚氰胺和三聚氰酸对小鼠肝脏无明显损伤作用。且三聚氰胺和三聚氰酸混合物能诱导小鼠肝脏细胞发生凋亡,Bax和caspase-3参与了三聚氰胺和三聚氰酸诱导小鼠肝细胞凋亡的调控。
Objective:This study is to investigate the mixture of melamine and cyanuric acid toxicity injury in the mouse liver tissue, and to identify its mechanism, to round out its toxicity studies and a comprehensive and objective assessment of melamine and cyanuric acid toxicity for the production of safe animal feed to provide a theoretical reference.
Method:This experiment uses 25 to 30 days aged mice as test object.30 days after gavage melamine and cyanuric acid poisoning attack, broken cervical vertebrae were killed, eyes and take out the liver blood test as follows:Use conventional method for melamine and cyanuric acid on liver function; Detection of liver superoxide dismutase (SOD) activity and malondialdehyde (MDA) content; with production of pathological liver biopsy and pathological examination of liver biopsy pathological changes;using RT-PCR, regulation of apoptosis related genes Bax, Bcl-2, Caspase-3 expression; by TUNEL assay of liver cell apoptosis and whether the rate of change, and by immunohistochemical detection of melamine and three poly cyanate on the regulation of apoptosis related genes Bax, Bcl-2, Caspase-3 protein expression
Results:After injection, melamine and cyanuric acid were mixed each dose group there are different degrees of liver cell degeneration, necrosis, nuclear condensation, fragmentation and disappear, some serious or even necrosis. As the infection dosage gradually increasing the content of MDA increased, while SOD activity decreased. Liver function tests, each dose group ALT, AST activity with the mixture of melamine and cyanuric acid dose increased gradually, TP content decreased correspondingly. TUNEL test results show:the mixed dose groups have different levels of apoptosis, and with the concentration, the more obvious apoptosis. RT-PCR results showed that Bax and caspase-3 expression was significantly increased, Bcl-2 gene expression was decreased, mixed high-dose group compared with the control group were significantly lower OD values were significantly different (P <0.01).Immunohistochemistry results showed that:Bax and caspase-3 are involved in the melamine and cyanuric acid induced apoptosis of liver cells, while Bcl-2 has not involved with melamine and cyanuric acid induced liver cell.
Conclusion:Mixture of melamine and cyanuric acid on liver injury in mice, with the mixture showed a definite increase in dose effect relationship, the higher the dose the more serious injury. The separate melamine and cyanuric acid in the mouse liver without significant injury. And the mixture of melamine and cyanuric acid can induce apoptosis in mouse liver cells, Bax and caspase-3 involved in the melamine and cyanuric acid induced apoptosis of liver cells.
研究方法：本实验采用25-30日龄小白鼠为试验对象,通过三聚氰胺及三聚氰酸灌胃攻毒30天后,断颈椎处死,眼眶采血并取出肝脏进行如下实验：采用常规方法检测三聚氰胺及三聚氰酸对肝功能的影响；检测肝脏组织中超氧化物歧化酶(SOD)的活性及丙二醛(MDA)的含量；采用制作病理组织切片法检查小鼠肝组织病理变化；采用RT-PCR法检测凋亡相关调控基因Bax、Bcl-2、Caspase-3的表达；采用TUNEL法检测肝脏细胞是否凋亡及凋亡率的变化,并采用免疫组织化学法检测三聚氰胺及三聚氰酸对凋亡相关调控基因Bax、Bcl-2、Caspase-3蛋白表达的影响。
研究结果：攻毒后,三聚氰胺和三聚氰酸混合各剂量组小鼠均出现不同程度的肝细胞变性、坏死,细胞核浓缩、碎裂、消失,有些严重甚至出现坏死灶。随着攻毒剂量的增加MDA的含量逐渐升高,而SOD活性降低。肝功能检测中,各剂量组ALT、AST活力随着三聚氰胺和三聚氰酸混合物剂量的增加而逐渐升高,TP含量随之而降低。TUNEL法检测结果显示：各混合剂量组均有不同程度的凋亡,且随着浓度升高,细胞凋亡越明显。RT-PCR结果显示Bax和caspase-3基因表达水平明显上升,Bcl-2基因表达水平明显下降,混合高剂量组与对照组比较OD值均明显降低差异极显著(P<0.01)。免疫组化结果显示：Bax和caspase-3都参与了三聚氰胺和三聚氰酸诱导小鼠肝细胞凋亡的调控,而Bcl-2与三聚氰胺和三聚氰酸诱导小鼠肝细胞凋亡无关。
研究结论：三聚氰胺和三聚氰酸混合物对小鼠肝脏有损伤作用,随着混合物剂量的上升呈一定的量效关系,剂量越高损伤情况越严重。而单独的三聚氰胺和三聚氰酸对小鼠肝脏无明显损伤作用。且三聚氰胺和三聚氰酸混合物能诱导小鼠肝脏细胞发生凋亡,Bax和caspase-3参与了三聚氰胺和三聚氰酸诱导小鼠肝细胞凋亡的调控。
Objective:This study is to investigate the mixture of melamine and cyanuric acid toxicity injury in the mouse liver tissue, and to identify its mechanism, to round out its toxicity studies and a comprehensive and objective assessment of melamine and cyanuric acid toxicity for the production of safe animal feed to provide a theoretical reference.
Method:This experiment uses 25 to 30 days aged mice as test object.30 days after gavage melamine and cyanuric acid poisoning attack, broken cervical vertebrae were killed, eyes and take out the liver blood test as follows:Use conventional method for melamine and cyanuric acid on liver function; Detection of liver superoxide dismutase (SOD) activity and malondialdehyde (MDA) content; with production of pathological liver biopsy and pathological examination of liver biopsy pathological changes;using RT-PCR, regulation of apoptosis related genes Bax, Bcl-2, Caspase-3 expression; by TUNEL assay of liver cell apoptosis and whether the rate of change, and by immunohistochemical detection of melamine and three poly cyanate on the regulation of apoptosis related genes Bax, Bcl-2, Caspase-3 protein expression
Results:After injection, melamine and cyanuric acid were mixed each dose group there are different degrees of liver cell degeneration, necrosis, nuclear condensation, fragmentation and disappear, some serious or even necrosis. As the infection dosage gradually increasing the content of MDA increased, while SOD activity decreased. Liver function tests, each dose group ALT, AST activity with the mixture of melamine and cyanuric acid dose increased gradually, TP content decreased correspondingly. TUNEL test results show:the mixed dose groups have different levels of apoptosis, and with the concentration, the more obvious apoptosis. RT-PCR results showed that Bax and caspase-3 expression was significantly increased, Bcl-2 gene expression was decreased, mixed high-dose group compared with the control group were significantly lower OD values were significantly different (P <0.01).Immunohistochemistry results showed that:Bax and caspase-3 are involved in the melamine and cyanuric acid induced apoptosis of liver cells, while Bcl-2 has not involved with melamine and cyanuric acid induced liver cell.
Conclusion:Mixture of melamine and cyanuric acid on liver injury in mice, with the mixture showed a definite increase in dose effect relationship, the higher the dose the more serious injury. The separate melamine and cyanuric acid in the mouse liver without significant injury. And the mixture of melamine and cyanuric acid can induce apoptosis in mouse liver cells, Bax and caspase-3 involved in the melamine and cyanuric acid induced apoptosis of liver cells.
引文
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