丙烯酰胺致小鼠睾丸组织的毒性作用及其CK18与Fas、FasL和Caspase-3表达的研究
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摘要
实验目的建立AA亚慢性染毒小鼠模型,研究AA对小鼠睾丸组织的生殖毒性作用,以及AA染毒后细胞骨架CK18与细胞凋亡相关因子Fas、FasL和Caspase-3表达的变化及其相互关系,进而为研究生殖细胞凋亡通路及分子调控机制,以及探讨AA的生殖毒性机制提供实验依据。
实验内容与方法
将健康5-6周龄清洁级雄性昆明种小鼠40只,随机分为4组,每组10只。用不同剂量的AA(0、10、20、40mg/kg)对小鼠连续腹腔染毒5周,每周6天,其中对照组给予等体积蒸馏水。每日测量体重,于首次染毒AA后第36天处死小鼠,计算睾丸系数。采用组织病理学技术(HE染色),RT-PCR技术,免疫组织化学法,分别从组织学水平,mRNA水平,蛋白水平,分析AA对小鼠睾丸组织的生殖毒性作用、CK18与细胞凋亡相关因子Fas、FasL和Caspase-3表达的变化及其相互关系。
实验结果
1、AA染毒期间,小鼠体重、睾丸重量及其睾丸系数均呈下降趋势,与对照组相比有统计学差异(P<0.01)。
2、HE染色结果显示,10mg/kg组与正常对照组比较,小鼠睾丸曲细精管排列基本规则,各级生精细胞未见明显改变。20mg/kg和40mg/kg剂量组小鼠,曲细精管排列不规则,生精上皮层次减少,各级生精细胞减少,管腔内成熟精子减少。
3、RT-PCR及免疫组化结果显示,CK18mRNA及蛋白在正常小鼠睾丸组织有表达；20mg/kg和40mg/kg剂量组与正常对照组比较,CK18mRNA及蛋白表达均明显降低(P<0.01)。
4、小鼠睾丸组织Fas、FasL、Caspase-3的表达,对照组生精小管中有散在分布的个别阳性细胞,10mg/kg组阳性细胞不明显；20mg/kg组和40mg/kg组阳性细胞增多,IOD值较正常对照组均明显增多,且有统计学差异(P<0.01)。
结论
1、AA亚慢性染毒可致小鼠体重增长缓慢,睾丸系数降低。
2、AA亚慢性染毒可使小鼠睾丸结构发生病理性改变,成熟精子减少,生精功能降低。
3、AA致小鼠睾丸组织CK18mRNA及蛋白的表达显著减少。推测AA生殖毒性作用
的可能机制为AA染毒使CK18mRNA表达减少,从而导致CK18生成减少。
4、AA可诱导小鼠睾丸组织细胞凋亡相关因子Fas、FasL与Caspase-3表达增加,从而激活Fas/FasL凋亡通路。表明在AA生殖毒性中存在着Caspase-3的依赖性凋亡机制。
5、CK18mRNA和蛋白的表达与Fas、FasL、Caspase-3的表达均呈负相关,表明AA诱导睾丸Fas、FasL、Caspase-3表达增加与CK18表达减少有一定的相关性。
Objective
This subject established a mice model of AA subchronic exposure, based on which, reproductive toxicity of AA in the testicular tissue, as well as the expression changes and interrelation with CK18and apoptosis related factors for Fas、FasL and Caspase-3were studied, so as to explore the molecular mechanisms and apoptosis pathways, and provide the experimental basis for germ cell apoptosis pathway and molecular regulation mechanism, as well as the mechanism of the AA reproductive toxicity.
Methods
Health KM mice for5-6weeks randomly divided into4groups, each group for10. Different doses of AA (0,10,20,40mg/kg) peritoneal injection for5weeks, each week for6days, the control group was given equal volume distilled water. The body weight was measured each day. After36days, the mice were killed. Testicular coefficients were calculated, then using histopathological technology (HE dyeing), RT-PCR technology, immunohistochemistry methods respectively detected the expression of CK18and apoptosis related factors for FasL, Caspase-3and Fas on the tissue, mRNA, protein level. We analysis the correlations between CK18and apoptosis related factors finally.
Result
1. During the acrylamide exposure, the body weight, testicles weight and organs coefficient were decreased, compared with the control, there was statistically significant (p<0.01).
2. Compared with the normal control group, HE staining showed that mice testis seminiferous tubes were basic rules, and no significant changes in spermatogenic cells at all levels in the10mg/kg group.20mg/kg group and40mg/kg seminiferous tubes are not in the basic rules, seminiferous epithelium level and all levels of spermatogenic cells depressed, mature sperm within the lumen also depressed in tubes.
3. RT-PCR and immunohistochemistry staining showed that CK18mRNA and protein expression of20mg/kg group and40mg/kg group in mouse testis were significantly decreased compared with the normal control group (P<0.01).
4. The expression of Fas, FasL, Caspase-3in the testicular tissue scattered a few positive cells in the control group, with10mg/kg group positive cells is not obvious,20mg/kg group and40mg/kg group more positive cells, of which, the IOD value was significantly increased, and statistically significant (P<0.01), compared with the normal control group.
Conclusion
1. The AA subchronic exposure resulted in the slow weight growth, with testicular coefficient reducing.
2. Subchronic exposure to AA can make the pathological changes of the mouse testis structure, reduction of the mature sperm as well as spermatogenic function.
3. The expression of CK18in gene and protein level decreased significantly, suggesting the changes of CK18expression may involved in the mechanism of the reproductive toxicity induced by AA.
4. The expression of apoptosis related factors for FasL Fas and Caspase-3induced by AA in the mice testicular tissue cell increased, resulting in activating the Fas/FasL apoptosis pathways. Show that the existence of caspase-3-dependent mechanism of apoptosis in AA reproductive toxicity.
5. The expression of CK18in the mRNA and protein levels were negatively correlated with the expression of Fas, FasL, Caspase-3, which was expression of the CK18in the mRNA and protein levels downregulated, but the expression of Fas, FasL, Caspase-3upregulated, indicating a correlation between the low expression of CK18and high expression of Fas, FasL, Caspase-3induced by AA in the testicular tissues.
实验内容与方法
将健康5-6周龄清洁级雄性昆明种小鼠40只,随机分为4组,每组10只。用不同剂量的AA(0、10、20、40mg/kg)对小鼠连续腹腔染毒5周,每周6天,其中对照组给予等体积蒸馏水。每日测量体重,于首次染毒AA后第36天处死小鼠,计算睾丸系数。采用组织病理学技术(HE染色),RT-PCR技术,免疫组织化学法,分别从组织学水平,mRNA水平,蛋白水平,分析AA对小鼠睾丸组织的生殖毒性作用、CK18与细胞凋亡相关因子Fas、FasL和Caspase-3表达的变化及其相互关系。
实验结果
1、AA染毒期间,小鼠体重、睾丸重量及其睾丸系数均呈下降趋势,与对照组相比有统计学差异(P<0.01)。
2、HE染色结果显示,10mg/kg组与正常对照组比较,小鼠睾丸曲细精管排列基本规则,各级生精细胞未见明显改变。20mg/kg和40mg/kg剂量组小鼠,曲细精管排列不规则,生精上皮层次减少,各级生精细胞减少,管腔内成熟精子减少。
3、RT-PCR及免疫组化结果显示,CK18mRNA及蛋白在正常小鼠睾丸组织有表达；20mg/kg和40mg/kg剂量组与正常对照组比较,CK18mRNA及蛋白表达均明显降低(P<0.01)。
4、小鼠睾丸组织Fas、FasL、Caspase-3的表达,对照组生精小管中有散在分布的个别阳性细胞,10mg/kg组阳性细胞不明显；20mg/kg组和40mg/kg组阳性细胞增多,IOD值较正常对照组均明显增多,且有统计学差异(P<0.01)。
结论
1、AA亚慢性染毒可致小鼠体重增长缓慢,睾丸系数降低。
2、AA亚慢性染毒可使小鼠睾丸结构发生病理性改变,成熟精子减少,生精功能降低。
3、AA致小鼠睾丸组织CK18mRNA及蛋白的表达显著减少。推测AA生殖毒性作用
的可能机制为AA染毒使CK18mRNA表达减少,从而导致CK18生成减少。
4、AA可诱导小鼠睾丸组织细胞凋亡相关因子Fas、FasL与Caspase-3表达增加,从而激活Fas/FasL凋亡通路。表明在AA生殖毒性中存在着Caspase-3的依赖性凋亡机制。
5、CK18mRNA和蛋白的表达与Fas、FasL、Caspase-3的表达均呈负相关,表明AA诱导睾丸Fas、FasL、Caspase-3表达增加与CK18表达减少有一定的相关性。
Objective
This subject established a mice model of AA subchronic exposure, based on which, reproductive toxicity of AA in the testicular tissue, as well as the expression changes and interrelation with CK18and apoptosis related factors for Fas、FasL and Caspase-3were studied, so as to explore the molecular mechanisms and apoptosis pathways, and provide the experimental basis for germ cell apoptosis pathway and molecular regulation mechanism, as well as the mechanism of the AA reproductive toxicity.
Methods
Health KM mice for5-6weeks randomly divided into4groups, each group for10. Different doses of AA (0,10,20,40mg/kg) peritoneal injection for5weeks, each week for6days, the control group was given equal volume distilled water. The body weight was measured each day. After36days, the mice were killed. Testicular coefficients were calculated, then using histopathological technology (HE dyeing), RT-PCR technology, immunohistochemistry methods respectively detected the expression of CK18and apoptosis related factors for FasL, Caspase-3and Fas on the tissue, mRNA, protein level. We analysis the correlations between CK18and apoptosis related factors finally.
Result
1. During the acrylamide exposure, the body weight, testicles weight and organs coefficient were decreased, compared with the control, there was statistically significant (p<0.01).
2. Compared with the normal control group, HE staining showed that mice testis seminiferous tubes were basic rules, and no significant changes in spermatogenic cells at all levels in the10mg/kg group.20mg/kg group and40mg/kg seminiferous tubes are not in the basic rules, seminiferous epithelium level and all levels of spermatogenic cells depressed, mature sperm within the lumen also depressed in tubes.
3. RT-PCR and immunohistochemistry staining showed that CK18mRNA and protein expression of20mg/kg group and40mg/kg group in mouse testis were significantly decreased compared with the normal control group (P<0.01).
4. The expression of Fas, FasL, Caspase-3in the testicular tissue scattered a few positive cells in the control group, with10mg/kg group positive cells is not obvious,20mg/kg group and40mg/kg group more positive cells, of which, the IOD value was significantly increased, and statistically significant (P<0.01), compared with the normal control group.
Conclusion
1. The AA subchronic exposure resulted in the slow weight growth, with testicular coefficient reducing.
2. Subchronic exposure to AA can make the pathological changes of the mouse testis structure, reduction of the mature sperm as well as spermatogenic function.
3. The expression of CK18in gene and protein level decreased significantly, suggesting the changes of CK18expression may involved in the mechanism of the reproductive toxicity induced by AA.
4. The expression of apoptosis related factors for FasL Fas and Caspase-3induced by AA in the mice testicular tissue cell increased, resulting in activating the Fas/FasL apoptosis pathways. Show that the existence of caspase-3-dependent mechanism of apoptosis in AA reproductive toxicity.
5. The expression of CK18in the mRNA and protein levels were negatively correlated with the expression of Fas, FasL, Caspase-3, which was expression of the CK18in the mRNA and protein levels downregulated, but the expression of Fas, FasL, Caspase-3upregulated, indicating a correlation between the low expression of CK18and high expression of Fas, FasL, Caspase-3induced by AA in the testicular tissues.
引文
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