亚硝酸盐暴露致雄性小鼠生殖毒性的研究

发布时间：2018-03-25 10:45

本文选题：亚硝酸盐暴露　切入点：生殖毒性　出处：《郑州大学》2017年硕士论文

【摘要】：亚硝酸盐作为一种常见的防腐剂和腌制剂,普遍应用于各种食品中,与人们的日常生活息息相关[1]。流行病学和大量的动物实验证明亚硝酸盐是一种毒物,急性中毒可导致高铁血红蛋白血症[2],慢性中毒则可致畸[3],致癌[4]。目前对亚硝酸盐的研究热点主要集中在癌症及神经毒性方面[4,5,6],但对其生殖毒性研究较少且缺乏亚慢性动物实验,本课题组在探讨亚硝酸盐暴露对小鼠神经系统毒性时发现其具有不孕效应[6,7],表明亚硝酸盐具有生殖毒性,因此,亚硝酸盐的雄性生殖毒性则引起了课题组的关注。哺乳动物精子的发生是一个高度复杂而又连续的过程,除了受内定基因表达的调控,还受表观遗传学的调控[8]。表观遗传学是研究不涉及DNA序列改变的可遗传修饰,包括DNA甲基化和组蛋白修饰等,它是生命活动的重要调控方式,也是近年来生殖医学领域研究的热点[9],参与所有的真核生物基因的表达过程,是生殖细胞正常增殖、分裂、分化的必须保障[9,10]。研究表明,表观遗传学异常可以引起精子发育障碍,与男性不育有着明确的联系[11],并且还与许多疾病的发生有关[12,13]。已有研究证实:外源性化合物如重金属镉、汞、砷,双氯酚A,吸烟、饮酒等依赖性行为均可引起机体表观遗传学改变[14,15],亚硝酸盐作为环境中最常见的无机化合物,对雄性生殖系统的影响机制目前还不甚明了,尤其在表观遗传学方面。目的建立亚硝酸盐暴露模型,通过观察小鼠睾丸生精细胞的增殖与凋亡情况及DNA甲基化和组蛋白去乙酰化相关酶的表达情况,初步探讨表观遗传学是否参与了亚硝酸盐暴露对雄性生殖系统的损伤过程,进而阐述亚硝酸盐暴露对雄性生殖毒性的表观遗传学机制,为辅助生殖技术中出现的表观遗传学问题及不孕不育的治疗进展提供新的思路。方法选取2月龄健康雄性昆明小鼠60只,随机分为三组:对照组(0.9%氯化钠溶液组)、低剂量亚硝酸钠溶液组(60mg/kg)、高剂量亚硝酸钠溶液组(120mg/kg),每组20只每日灌胃一次,连续3个月。观察各组小鼠的生长发育状况,采用HE染色法观察小鼠睾丸组织病理形态学改变,BrdU法标记细胞增殖,TUNEL法检测细胞凋亡。免疫荧光和Western blot法分析检测各组小鼠睾丸组织炎症相关蛋白iNOS、COX2、NF-κB-p65的表达情况,凋亡蛋白CytochromeC(Cytc)和Caspase3的表达,DNA甲基化相关酶、组蛋白去乙酰化相关酶的表达。结果与对照组相比,亚硝酸盐暴露组小鼠体重增加缓慢,睾丸及附睾脏器指数降低(P0.01);HE染色结果显示亚硝酸盐暴露组小鼠睾丸组织生精细胞排列紊乱,生精细胞脱落、曲细精管变形,形态发生病理性改变;BrdU结果显示亚硝酸盐暴露组小鼠睾丸组织细胞增殖较对照组明显减少、TUNEL结果显示亚硝酸盐暴露组小鼠睾丸组织细胞凋亡较对照组明显增加(P0.01);免疫荧光和Western blot结果显示iNOS、COX2、NF-κB-p65、Cytc和Caspase3的表达明显多于对照组,同时DNA甲基化相关酶及组蛋白去乙酰化相关酶的表达均比对照组增多(P0.01),以上结果均呈现剂量依赖性。结论亚硝酸盐暴露通过抑制小鼠生长发育及睾丸生精细胞增殖,诱导睾丸生精细胞氧化应激损伤及凋亡,造成雄性生殖毒性;DNA甲基化及组蛋白去乙酰化水平的升高,提示表观遗传学的改变可能参与了亚硝酸盐暴露对雄性生殖系统的损伤过程及调控机制。
[Abstract]:Nitrite as a common preservative and curing agent, widely used in all kinds of food, and people's daily life is closely related to the epidemiology of [1]. and a large number of animal experiments showed that nitrite is a kind of poison, acute poisoning can cause methemoglobinemia [2], chronic poisoning can be teratogenic carcinogenic [3], [4]. hotspot of nitrite mainly concentration of [4,5,6] in cancer and neurotoxicity, but the study of reproductive toxicity is less and lack of subchronic animal experiments, the research group in the study of nitrite exposure on the nervous system of mice and found that it has the effect of [6,7] showed that the nitrite with infertility, reproductive toxicity, therefore, male reproductive toxicity of nitrite caused the group occurred attention. Mammalian sperm is a highly complex and continuous process, in addition to the regulation of gene expression by default Also, the epigenetic regulation of [8]. epigenetics is the study of genetic modification does not involve a change in DNA sequence, including DNA methylation and histone modification, it is an important regulation of life activities, but also the research focus of [9] in the field of reproductive medicine in recent years, the expression process involved all eukaryotic genes that is the normal reproductive cell division, proliferation, differentiation of [9,10]. security research must show that epigenetic abnormalities can cause sperm development disorder, a clear link between [11] and male sterility, and also with many diseases related [12,13]. studies have confirmed that exogenous compounds such as cadmium, mercury, arsenic, Dichlorophen A smoking, drinking, etc. can cause the body dependent behavior of epigenetic changes in [14,15], nitrite as the most common inorganic compounds in the environment, the influence mechanism of the male reproductive system at present Not very clear, especially in epigenetics. Objective to establish a model of nitrite exposure, the expression of spermatogenic cells in testis of mice by observing the proliferation and apoptosis and DNA methylation and histone deacetylase enzymes, preliminary discussion table is involved in the damage process of nitrite exposure on male reproductive system genetics concept, and this nitrite exposure on male reproductive toxicity of epigenetic mechanisms, to provide new ideas for treatment of assisted reproductive technology in epigenetics and infertility. 2 month old healthy male Kunming mice 60 methods, were randomly divided into three groups: control group (0.9% Sodium Chloride Solution group), low dose of sodium nitrite the solution group (60mg/kg), high dose of sodium nitrite solution (120mg/kg group), 20 rats in each group by gavage once daily, for 3 months. The growth and development status of mice were observed, HE was used to observe the change of testis pathological staining and BrdU labeling of cell proliferation, cell apoptosis was detected by TUNEL. Immunofluorescence and Western blot detection and analysis of related protein of mice testis tissue inflammation, iNOS, COX2, expression of NF- K B-p65, apoptosis protein CytochromeC (Cytc) and the expression of Caspase3, DNA methyl the expression of enzymes involved in histone deacetylase enzymes. Results compared with the control group, nitrite exposure group mice body weight increased slowly, the testis and epididymal index decreased (P0.01); HE staining showed that the nitrite exposure of the testis tissue in mice spermatogenic cell disorder, spermatogenic cell shed, seminiferous tube deformation morphological, pathological change; BrdU results showed that nitrite exposure on testis cell proliferation were significantly reduced compared with the control group, TUNEL results showed that the nitrite exposure of mice testis Cell apoptosis pill significantly increased compared with the control group (P0.01); immunofluorescence and Western blot showed that iNOS, COX2, NF- K B-p65, expression of Cytc and Caspase3 were significantly more than the control group, the expression of DNA methylation related enzymes and histone deacetylase enzymes were higher than control group (P0.01), above the results were dose-dependent. Conclusion nitrite exposure by inhibiting the growth and proliferation of spermatogenic cells, induced testicular injury and apoptosis of spermatogenic cells caused by oxidative stress, male reproductive toxicity; increased DNA methylation and histone acetylation levels, suggesting that epigenetic changes may be involved in the nitrite exposure injury the process and mechanisms of the male reproductive system.

【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R114

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