本文選題：亞硝酸鹽暴露　切入點(diǎn)：生殖毒性　出處：《鄭州大學(xué)》2017年碩士論文

【摘要】：亞硝酸鹽作為一種常見的防腐劑和腌制劑,普遍應(yīng)用于各種食品中,與人們的日常生活息息相關(guān)[1]。流行病學(xué)和大量的動(dòng)物實(shí)驗(yàn)證明亞硝酸鹽是一種毒物,急性中毒可導(dǎo)致高鐵血紅蛋白血癥[2],慢性中毒則可致畸[3],致癌[4]。目前對(duì)亞硝酸鹽的研究熱點(diǎn)主要集中在癌癥及神經(jīng)毒性方面[4,5,6],但對(duì)其生殖毒性研究較少且缺乏亞慢性動(dòng)物實(shí)驗(yàn),本課題組在探討亞硝酸鹽暴露對(duì)小鼠神經(jīng)系統(tǒng)毒性時(shí)發(fā)現(xiàn)其具有不孕效應(yīng)[6,7],表明亞硝酸鹽具有生殖毒性,因此,亞硝酸鹽的雄性生殖毒性則引起了課題組的關(guān)注。哺乳動(dòng)物精子的發(fā)生是一個(gè)高度復(fù)雜而又連續(xù)的過程,除了受內(nèi)定基因表達(dá)的調(diào)控,還受表觀遺傳學(xué)的調(diào)控[8]。表觀遺傳學(xué)是研究不涉及DNA序列改變的可遺傳修飾,包括DNA甲基化和組蛋白修飾等,它是生命活動(dòng)的重要調(diào)控方式,也是近年來生殖醫(yī)學(xué)領(lǐng)域研究的熱點(diǎn)[9],參與所有的真核生物基因的表達(dá)過程,是生殖細(xì)胞正常增殖、分裂、分化的必須保障[9,10]。研究表明,表觀遺傳學(xué)異�？梢砸鹁影l(fā)育障礙,與男性不育有著明確的聯(lián)系[11],并且還與許多疾病的發(fā)生有關(guān)[12,13]。已有研究證實(shí):外源性化合物如重金屬鎘、汞、砷,雙氯酚A,吸煙、飲酒等依賴性行為均可引起機(jī)體表觀遺傳學(xué)改變[14,15],亞硝酸鹽作為環(huán)境中最常見的無機(jī)化合物,對(duì)雄性生殖系統(tǒng)的影響機(jī)制目前還不甚明了,尤其在表觀遺傳學(xué)方面。目的建立亞硝酸鹽暴露模型,通過觀察小鼠睪丸生精細(xì)胞的增殖與凋亡情況及DNA甲基化和組蛋白去乙�；嚓P(guān)酶的表達(dá)情況,初步探討表觀遺傳學(xué)是否參與了亞硝酸鹽暴露對(duì)雄性生殖系統(tǒng)的損傷過程,進(jìn)而闡述亞硝酸鹽暴露對(duì)雄性生殖毒性的表觀遺傳學(xué)機(jī)制,為輔助生殖技術(shù)中出現(xiàn)的表觀遺傳學(xué)問題及不孕不育的治療進(jìn)展提供新的思路。方法選取2月齡健康雄性昆明小鼠60只,隨機(jī)分為三組:對(duì)照組(0.9%氯化鈉溶液組)、低劑量亞硝酸鈉溶液組(60mg/kg)、高劑量亞硝酸鈉溶液組(120mg/kg),每組20只每日灌胃一次,連續(xù)3個(gè)月。觀察各組小鼠的生長發(fā)育狀況,采用HE染色法觀察小鼠睪丸組織病理形態(tài)學(xué)改變,BrdU法標(biāo)記細(xì)胞增殖,TUNEL法檢測細(xì)胞凋亡。免疫熒光和Western blot法分析檢測各組小鼠睪丸組織炎癥相關(guān)蛋白iNOS、COX2、NF-κB-p65的表達(dá)情況,凋亡蛋白CytochromeC(Cytc)和Caspase3的表達(dá),DNA甲基化相關(guān)酶、組蛋白去乙酰化相關(guān)酶的表達(dá)。結(jié)果與對(duì)照組相比,亞硝酸鹽暴露組小鼠體重增加緩慢,睪丸及附睪臟器指數(shù)降低(P0.01);HE染色結(jié)果顯示亞硝酸鹽暴露組小鼠睪丸組織生精細(xì)胞排列紊亂,生精細(xì)胞脫落、曲細(xì)精管變形,形態(tài)發(fā)生病理性改變;BrdU結(jié)果顯示亞硝酸鹽暴露組小鼠睪丸組織細(xì)胞增殖較對(duì)照組明顯減少、TUNEL結(jié)果顯示亞硝酸鹽暴露組小鼠睪丸組織細(xì)胞凋亡較對(duì)照組明顯增加(P0.01);免疫熒光和Western blot結(jié)果顯示iNOS、COX2、NF-κB-p65、Cytc和Caspase3的表達(dá)明顯多于對(duì)照組,同時(shí)DNA甲基化相關(guān)酶及組蛋白去乙�；嚓P(guān)酶的表達(dá)均比對(duì)照組增多(P0.01),以上結(jié)果均呈現(xiàn)劑量依賴性。結(jié)論亞硝酸鹽暴露通過抑制小鼠生長發(fā)育及睪丸生精細(xì)胞增殖,誘導(dǎo)睪丸生精細(xì)胞氧化應(yīng)激損傷及凋亡,造成雄性生殖毒性;DNA甲基化及組蛋白去乙�；降纳�,提示表觀遺傳學(xué)的改變可能參與了亞硝酸鹽暴露對(duì)雄性生殖系統(tǒng)的損傷過程及調(diào)控機(jī)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.

【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R114

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