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

发布时间：2018-05-27 10:47

本文选题：丙烯酰胺 + 小鼠睾丸　；参考：《山西医科大学》2012年硕士论文

【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(P0.01)。 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(P0.01)。 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(P0.01)。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。
[Abstract]:Objective to study the reproductive toxicity of AA to testis and the relationship between the expression of cytoskeleton CK18 and apoptosis-related factors FasL and Caspase-3. It provides experimental basis for studying germ cell apoptosis pathway, molecular regulation mechanism and the mechanism of reproductive toxicity of AA. Experimental contents and methods Forty male Kunming mice of 5-6 weeks old were randomly divided into 4 groups with 10 mice in each group. The mice were exposed to different doses of AAAZO 1010 ~ 20g / kg for 5 weeks, 6 days a week, and the control group was given distilled water of the same volume. The mice were killed on the 36th day after the first AA exposure and the testicular coefficient was calculated. By using histopathological technique, HE staining and RT-PCR, immunohistochemical method, mRNA and protein levels were determined, respectively. To analyze the reproductive toxicity of AA on testis of mice and the relationship between CK18 and the expression of FasL and Caspase-3 in apoptosis-related cytokines. Experimental results (1) the weight, testis weight and testicular coefficient of the mice decreased during the exposure to AA, and there was a significant difference compared with the control group (P 0.01). 2the results of HE staining showed that the basic rules of seminiferous tubules arrangement were observed in the 10 mg / kg group compared with the normal control group. The seminiferous cells of all levels of spermatogenic cells did not change significantly in the groups of .20mg / kg and 40mg/kg dosage. The seminiferous tubules arranged irregularly and the layers of seminiferous epithelium decreased. The number of spermatogenic cells and mature spermatozoa in the lumen decreased. 3The results of RT-PCR and immunohistochemistry showed that the expression of CK18 mRNA and protein in testis of normal mice was 20 mg / kg and the expression of CK18 mRNA and protein in 40mg/kg group was significantly lower than that in normal control group (P 0.01). (4) the expression of Fas-FasL- Caspase-3 in testicular tissues of mice, the positive cells in the seminiferous tubules of the control group were not significantly increased in 10 mg / kg group and 20 mg / kg group and 40mg/kg group were significantly higher than those in the normal control group, and there was a statistical difference between them (P0.01). Conclusion 1the weight gain and testis coefficient decreased after subchronic exposure to AA. 2Subchronic exposure to AA could induce pathological changes of testis structure, decrease of mature spermatozoa and decrease of spermatogenic function in mice. 3The expression of CK18mRNA and protein in testis of mice induced by AA was significantly decreased. Hypothetical reproductive toxicity of AA The possible mechanism is that AA exposure reduces the expression of CK18mRNA, resulting in reduced CK18 production. 4the expression of Fas-FasL and Caspase-3 in mouse testicular tissue was increased by AA-induced apoptosis, thus activating the apoptosis pathway of Fas/FasL. It is suggested that there is a mechanism of Caspase-3 dependent apoptosis in AA reproductive toxicity. 5The expression of CK18 mRNA and protein was negatively correlated with the expression of Fas-FasL- Caspase-3, which indicated that the increased expression of Caspase-3 in Fas-FasLL was correlated with the decrease of CK18 expression in the testis induced by AA.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R114

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