發(fā)布時(shí)間：2018-06-09 04:50

  本文選題：二硫化碳 + 氧化應(yīng)激��； 參考：《山東大學(xué)》2014年碩士論文

【摘要】：研究背景 二硫化碳(Carbon disulfide, CS2)是工業(yè)上常用的有機(jī)溶劑和化工原料,廣泛用于粘膠纖維、玻璃紙、橡膠等的生產(chǎn)過程中。CS2具有多系統(tǒng)毒性,長期低劑量暴露于CS2可導(dǎo)致神經(jīng)、心血管、內(nèi)分泌系統(tǒng)等的病變。此外,值得關(guān)注的是CS2具有明顯的生殖損傷毒作用,可導(dǎo)致男性睪丸萎縮、精子質(zhì)量下降、性功能減退、性激素分泌紊亂等,并可導(dǎo)致女性月經(jīng)周期紊亂、自然流產(chǎn)、早產(chǎn)等。本課題組通過前瞻性隊(duì)列研究發(fā)現(xiàn),暴露組女工早早孕丟失率顯著高于對(duì)照組,并且暴露組女工的妊娠時(shí)間與對(duì)照組相比明顯延長,說明CS2影響胚胎的著床過程。動(dòng)物實(shí)驗(yàn)研究發(fā)現(xiàn),小鼠在圍植入期暴露于CS2導(dǎo)致胚胎植入數(shù)目明顯減少,但其毒性作用機(jī)制尚未明確。 氧化應(yīng)激可以導(dǎo)致脂質(zhì)過氧化、DNA損傷及蛋白質(zhì)結(jié)構(gòu)和功能的改變。研究表明,接觸CS2可引起機(jī)體氧化應(yīng)激反應(yīng),機(jī)體處于高氧化應(yīng)激水平可增加自然流產(chǎn)的風(fēng)險(xiǎn)。提示孕期CS2暴露可以通過改變機(jī)體的氧化應(yīng)激水平而影響胚胎正常著床。課題組前期實(shí)驗(yàn)結(jié)果顯示,胚胎植入期暴露于CS2可導(dǎo)致孕鼠子宮內(nèi)膜細(xì)胞DNA損傷,但與氧化應(yīng)激的關(guān)系尚不明確。DNA甲基化可以影響基因轉(zhuǎn)錄過程調(diào)節(jié)植入相關(guān)蛋白的表達(dá)水平,影響胚胎植入。DNA甲基化在調(diào)節(jié)基因功能過程中需要甲基轉(zhuǎn)移酶(DNA methyltransferase, DNMT)的參與。Logan等研究發(fā)現(xiàn),低甲基化水平可以增強(qiáng)子宮內(nèi)膜對(duì)胚胎的容受性,有利于胚胎植入。氧化應(yīng)激可引起機(jī)體DNA甲基化水平的改變,同時(shí)氧化應(yīng)激導(dǎo)致的DNA氧化損傷可啟動(dòng)機(jī)體的堿基切除修復(fù)過程,而參與堿基切除修復(fù)的有關(guān)酶需要經(jīng)過甲基化后才能發(fā)揮作用。綜上,我們推測在DNA氧化損傷后,需要DNMT的高表達(dá)以啟動(dòng)DNA損傷修復(fù)過程。因此,我們假設(shè)在孕鼠暴露于CS2后,氧化應(yīng)激、DNA損傷和DNA甲基化的單獨(dú)或聯(lián)合作用是胚胎植入障礙的重要機(jī)制。 本研究擬運(yùn)用不同胚胎植入時(shí)段CS2暴露與胚胎植入障礙關(guān)系的時(shí)間依賴動(dòng)物模型,檢測暴露后不同染毒時(shí)間點(diǎn)和觀察終點(diǎn)孕鼠子宮組織的氧化應(yīng)激、子宮內(nèi)膜細(xì)胞DNA損傷、DNA氧化損傷生物標(biāo)志物8-OH-dG以及DNMT表達(dá)水平,探討氧化應(yīng)激、DNA損傷及DNA甲基化在CS2致胚胎植入障礙中的毒性作用機(jī)制。 研究目的 構(gòu)建胚胎植入期不同時(shí)點(diǎn)暴露于CS2致胚胎植入障礙動(dòng)物模型,檢測不同染毒時(shí)間點(diǎn)與不同觀察終點(diǎn)的孕鼠子宮組織氧化應(yīng)激、子宮內(nèi)膜細(xì)胞DNA損傷、8-OH-dG以及DNMT表達(dá)水平,分析氧化應(yīng)激、DNA損傷和DNA甲基化在CS2所致的胚胎植入障礙中的作用及其致病機(jī)制,進(jìn)一步加深對(duì)圍植入期CS2暴露所致胚胎植入障礙毒作用機(jī)制的了解,為環(huán)境因素生殖損傷研究提供可借鑒的理論依據(jù)和研究方法。 研究方法 1.研究設(shè)計(jì) 研究設(shè)計(jì)分為三部分。第一部分用于建立CS2暴露致胚胎植入障礙時(shí)間依賴動(dòng)物模型,通過胚胎植入障礙現(xiàn)象尋找CS2致胚胎植入障礙敏感暴露時(shí)間點(diǎn),并檢測孕鼠子宮組織氧化應(yīng)激水平,分析氧化應(yīng)激在CS2致胚胎植入障礙機(jī)制中的作用。第二部分在第一部分實(shí)驗(yàn)的基礎(chǔ)上,選擇CS2致胚胎植入障礙敏感時(shí)點(diǎn)使孕鼠暴露于CS2,并在不同時(shí)點(diǎn)終止實(shí)驗(yàn),檢測孕鼠子宮內(nèi)膜細(xì)胞DNA損傷和子宮組織8-OH-dG水平,分析在CS2致胚胎植入障礙毒作用機(jī)制中DNA損傷的作用及DNA損傷與氧化應(yīng)激的關(guān)系。第三部分動(dòng)物處理同第二部分,檢測孕鼠子宮組織中DNMT表達(dá)水平,分析在CS2致胚胎植入障礙毒作用機(jī)制中DNA甲基化的作用。 2.暴露時(shí)間 本實(shí)驗(yàn)根據(jù)C82暴露時(shí)間的不同分為4種處理,即動(dòng)物分別在孕3d、孕4d、孕5d和孕6d(記為GD3、GD4、GD5和GD6)接受一次處理(C82或溶劑)。 3.觀察終點(diǎn) 選取暴露后的連續(xù)時(shí)間點(diǎn)作為觀察終點(diǎn),四種處理共有26個(gè)觀察終點(diǎn),即第一個(gè)處理組分別在暴露后第6h、12h、18h、24h、48h、72h、96h和144h(即GD9)共設(shè)有8個(gè)觀察終點(diǎn)；第二個(gè)處理組分別在暴露后6h、12h、18h、24h、48h、72h和120h(即GD9)共設(shè)有7個(gè)觀察終點(diǎn)；第三個(gè)處理組分別在暴露后6h、12h、18h、24h、48h和96h(即GD9)共設(shè)有6個(gè)觀察終點(diǎn)；第四個(gè)處理組分別在暴露后6h、12h、18h、24h和72h(即GD9)共設(shè)有5個(gè)觀察終點(diǎn)。 各處理組的最后1個(gè)觀察終點(diǎn)(GD9)用于觀察母體毒性及胚胎植入數(shù)目,其余各觀察終點(diǎn)用于檢測氧化應(yīng)激、DNA損傷及DNMT1表達(dá)水平等。 每個(gè)觀察終點(diǎn)包含1個(gè)CS2暴露組和1個(gè)溶劑對(duì)照組。 4.動(dòng)物受孕與分組 實(shí)驗(yàn)前清潔級(jí)性成熟昆明種小鼠適應(yīng)性喂養(yǎng)一周,按雌雄1：1合籠,次日晨檢查陰栓,陰栓陽性者記為孕第一天(記為GD1),隨機(jī)分到26個(gè)觀察終點(diǎn)。每個(gè)觀察終點(diǎn)組含12只孕鼠,再隨機(jī)分配進(jìn)入暴露組和對(duì)照組(各含6只孕鼠)。 5.孕鼠處理 孕鼠分別按照實(shí)驗(yàn)設(shè)計(jì)的暴露時(shí)點(diǎn)接受1次腹腔注射(CS2暴露劑量為631.4mg/kg,染毒前配置；對(duì)照組為橄欖油)；注射容量為0.1m1/10g體重。 6.樣本收集 4種處理組中最后一個(gè)觀察終點(diǎn)(GD9)的孕鼠在孕9d經(jīng)脫臼處死,取子宮、卵巢、肝、脾、腎和胚胎并稱重,計(jì)數(shù)胚胎植入數(shù)目；4種處理組中其他觀察終點(diǎn)的孕鼠在設(shè)計(jì)的觀察終點(diǎn)經(jīng)脫臼處死后取子宮組織,一半子宮組織經(jīng)手動(dòng)勻漿后取上清液,-80℃冰箱保存?zhèn)溆�。另一半子宮組織采用手工機(jī)械刮取法直接刮取孕鼠子宮內(nèi)膜細(xì)胞用于DNA損傷測定。 7.孕鼠子宮組織氧化應(yīng)激、DNA損傷、8-OH-dG和DNMT表達(dá)水平的測定 運(yùn)用考馬斯亮藍(lán)法對(duì)暴露組和對(duì)照組孕鼠子宮組織勻漿液進(jìn)行蛋白定量；運(yùn)用分光光度法測定孕鼠子宮組織勻漿液中丙二醛(MDA)、過氧化物歧化酶(SOD)、谷胱甘肽過氧化物酶(GSH-PX)和過氧化氫酶(CAT)水平；運(yùn)用酶聯(lián)免疫吸附測定法(enzymes linked immunosorbent assay, ELISA)測定孕鼠子宮組織中8-OH-dG含量；應(yīng)用彗星實(shí)驗(yàn)(comet assay)檢測孕鼠子宮內(nèi)膜細(xì)胞DNA損傷水平；運(yùn)用十二烷基磺酸鈉-聚丙烯酰胺凝膠電泳技術(shù)(SDS-PAGE)和蛋白免疫印跡法(western blotting)法測定孕鼠子宮組織中甲基轉(zhuǎn)移酶1(DNAmethyltransferase1, DNMT1)含量。 8.統(tǒng)計(jì)學(xué)分析 使用SPSS20.0進(jìn)行統(tǒng)計(jì)學(xué)分析,經(jīng)正態(tài)性檢驗(yàn),正態(tài)及近似正態(tài)分布的測量指標(biāo)均以x±s表示。首先對(duì)各指標(biāo)進(jìn)行方差齊性檢驗(yàn)：若方差齊,則采用單因素方差分析(ONE-WAY ANOVA)進(jìn)行F檢驗(yàn),同時(shí)采用Dunnett's t test進(jìn)行暴露組和對(duì)照組的比較；若方差不齊,則采用非參數(shù)統(tǒng)計(jì)方法(Kruskal-Wallis H法)。并運(yùn)用相關(guān)分析分析各指標(biāo)間的關(guān)系。統(tǒng)計(jì)學(xué)檢驗(yàn)水準(zhǔn)設(shè)定為α=0.05。 研究結(jié)果 1.圍植入期暴露于CS2對(duì)孕鼠母體及胚胎的毒性作用 母體毒性：孕鼠在胚胎植入不同時(shí)間點(diǎn)暴露于CS2后,孕鼠GD9體重和體重凈增量各暴露組與對(duì)照組比較,差異無統(tǒng)計(jì)學(xué)意義(P0.05)；孕鼠子宮、卵巢、肝、脾以及腎等臟器的重量及臟器系數(shù)組間比較,差異無統(tǒng)計(jì)學(xué)意義(P0.05)。說明該暴露劑量所致的母體毒性較小。 胚胎毒性：各暴露組胚胎植入數(shù)目均明顯低于對(duì)照組(P0.01)。GD3.GD4. GD5和GD6各暴露組的胚胎植入率分別為56.95%、36.26%、39.55%和52.74%,與對(duì)照組比較差異均有統(tǒng)計(jì)學(xué)意義(P0.01),暴露CS2后胚胎植入率明顯降低,GD4暴露組的胚胎植入率最低(P0.05)。在校正胚胎植入數(shù)目后胚胎均重與對(duì)照組比較,差異無統(tǒng)計(jì)學(xué)意義(P0.05)。 2.圍植入期暴露于CS2對(duì)孕鼠子宮組織氧化應(yīng)激的影響 CS2暴露后18h,各暴露組孕鼠子宮組織中MDA水平明顯升高,與對(duì)照組比較差異有統(tǒng)計(jì)學(xué)意義(P0.01),其中GD4暴露組孕鼠子宮組織MDA水平較對(duì)照組增高131.4%, GD3、GD5和GD6較對(duì)照組分別增高120.8%、121.6%和104.9%。此外,不同暴露時(shí)間點(diǎn)不同觀察終點(diǎn)孕鼠子宮組織中SOD、GSH-PX和CAT水平明顯降低,與對(duì)照組比較差異有統(tǒng)計(jì)學(xué)意義(P0.01):CAT水平在暴露后12h明顯降低(P0.01),GSH-PX水平在暴露后12h和18h明顯降低(P0.01), SOD水平在暴露后18h顯著降低(P0.01)。相關(guān)分析結(jié)果顯示,暴露后18h孕鼠子宮組織中MDA水平和GD9胚胎植入數(shù)目呈明顯負(fù)相關(guān)關(guān)系(r=-0.783,P0.01)。 3.圍植入期暴露于CS2對(duì)孕鼠子宮內(nèi)膜細(xì)胞DNA損傷的影響 孕鼠子宮內(nèi)膜細(xì)胞DNA損傷各項(xiàng)指標(biāo)(TL、TM、OTM和TDNA%)在CS2暴露后6h時(shí)明顯升高(P0.01),并在暴露后18h時(shí)達(dá)到最高點(diǎn)(P0.01),之后各指標(biāo)值逐漸下降。相關(guān)分析結(jié)果顯示,CS2暴露后18h時(shí)孕鼠子宮內(nèi)膜細(xì)胞TM、OTM、TL和TDNA%與胚胎植入數(shù)目之間呈明顯負(fù)相關(guān)關(guān)系(r=-0.804、-0.847、-0.934和-0.863,P0.01)。 4.圍植入期暴露于CS2對(duì)孕鼠子宮組織8-OH-dG水平的影響 與對(duì)照組比較,孕鼠CS2暴露后18h和24h時(shí)孕鼠子宮組織8-OH-dG水平顯著增高,差異具有統(tǒng)計(jì)學(xué)意義(P0.01)。在18h和24h時(shí)孕鼠子宮組織8-OH-dG水平與對(duì)照組比較,分別升高893.8%和647.4%(P0.01)。 5.圍植入期暴露于CS2對(duì)孕鼠子宮組織DNMT1水平的影響 孕鼠在GD4暴露于CS2后6h時(shí)孕鼠子宮組織DNMT1表達(dá)水平明顯降低,而在暴露后12h時(shí)孕鼠子宮組織DNMT1表達(dá)水平明顯升高,與對(duì)照組比較差異均有統(tǒng)計(jì)學(xué)意義(P0.01)。相關(guān)分析結(jié)果顯示,CS2暴露后12h孕鼠子宮組織DNMT1表達(dá)水平與胚胎植入數(shù)目之間呈明顯負(fù)相關(guān)關(guān)系(r=-0.433,P0.01)。 6.氧化應(yīng)激、DNA損傷和8-OH-dG水平與CS2暴露的關(guān)系 孕鼠GD4暴露于CS2后DNA損傷各項(xiàng)指標(biāo)6h時(shí)出現(xiàn)明顯改變,而暴露于CS2后18h時(shí)子宮組織MDA升高具有統(tǒng)計(jì)學(xué)意義,提示CS2所致孕鼠子宮組織DNA損傷改變?cè)缬谘趸瘧?yīng)激變化。相關(guān)分析結(jié)果顯示,作為DNA氧化損傷生物標(biāo)志物的8-OH-dG與DNA損傷指標(biāo)TDNA%、TL、TM和OTM之間呈正相關(guān)(r=0.766、0.688、0.738和0.771,P0.01),且在氧化應(yīng)激最高點(diǎn)時(shí)子宮內(nèi)膜細(xì)胞DNA損傷程度達(dá)到高峰,提示氧化應(yīng)激引起的DNA氧化損傷進(jìn)一步加重孕鼠子宮組織的DNA損傷程度。 結(jié)論 1.圍植入期不同時(shí)間暴露于CS2均可導(dǎo)致孕鼠子宮組織發(fā)生明顯且短暫的氧化應(yīng)激反應(yīng),呈現(xiàn)出明顯的時(shí)間變化規(guī)律且變化趨勢一致,與暴露時(shí)點(diǎn)無關(guān)。 2.圍植入期暴露于C82可導(dǎo)致孕鼠子宮內(nèi)膜細(xì)胞DNA損傷,并呈現(xiàn)出明顯的時(shí)間變化規(guī)律；氧化應(yīng)激通過引起DNA氧化損傷進(jìn)一步加重子宮內(nèi)膜細(xì)胞的DNA損傷程度。 3.子宮組織氧化應(yīng)激水平和內(nèi)膜細(xì)胞DNA損傷水平與CS2致胚胎植入障礙程度一致,兩者協(xié)同作用可能是CS2暴露致胚胎植入障礙的重要機(jī)制之一。 4.圍植入期暴露于CS2導(dǎo)致的子宮組織DNMT1表達(dá)水平的變化,可能與機(jī)體在氧化應(yīng)激和細(xì)胞DNA損傷毒性作用條件下對(duì)子宮內(nèi)膜容受性的調(diào)節(jié)機(jī)制有關(guān)。
[Abstract]:Research background
Carbon disulfide (CS2) is a common organic solvent and chemical raw material used in industry. It is widely used in the production of viscose fiber, glass paper and rubber, and.CS2 has multiple systemic toxicity. Long term low dose exposure to CS2 can lead to diseases such as nerve, cardiovascular, endocrine system and so on. In addition, it is important to pay attention to the obvious birth of CS2. The effect of colonization damage can cause male testicular atrophy, sperm quality decline, sexual dysfunction, sexual hormone secretion disorder and so on, and can lead to menstrual cycle disorder, spontaneous abortion, preterm birth and so on. Our group through prospective cohort study found that the loss rate of early pregnancy in exposed groups was significantly higher than that of the control group, and the pregnancy of women exposed groups was exposed to pregnancy. The time compared with the control group was significantly longer than that of the control group, indicating that CS2 affects the implantation process of the embryo. Animal experiments have found that the number of mice exposed to CS2 in the peri implantation period leads to a significant decrease in the number of embryo implantation, but the mechanism of its toxicity is not clear.
Oxidative stress can lead to lipid peroxidation, DNA damage and changes in protein structure and function. The study shows that exposure to CS2 can cause oxidative stress in the body, and the high oxidative stress level of the body can increase the risk of spontaneous abortion. It suggests that CS2 exposure during pregnancy can affect the normal implantation of embryos by changing the oxidative stress level of the body. The previous experimental results showed that exposure to CS2 in embryo implantation could lead to DNA damage in endometrial cells of pregnant mice, but the relationship with oxidative stress was not clear that.DNA methylation could affect the gene transcription process to regulate the expression of implantable protein, and the effect of.DNA methylation on the regulation of gene function in embryo implantation was necessary. The involvement of DNA methyltransferase (DNMT) in.Logan and other studies found that the level of low methylation can enhance the receptivity of the endometrium to the embryo, which is beneficial to the implantation of the embryo. Oxidative stress can cause the changes of the level of DNA methylation in the body, and the oxidative stress caused by oxidative stress can initiate the process of the base resection and repair of the body. The enzymes involved in base excision repair need to be methylation. To sum up, we speculate that after the DNA oxidative damage, the high expression of DNMT is needed to start the DNA damage repair process. Therefore, we assume that the pregnant rats are exposed to CS2, oxidative stress, the individual or combined effect of DNA damage and DNA methylation is an embryo implantation barrier. An important mechanism for hindrance.
The time dependent animal model of the relationship between CS2 exposure and embryo implantation barrier in different embryo implantation period was used to detect the oxidative stress, DNA damage of endometrium cells, 8-OH-dG and DNMT expression level of DNA oxidative damage, and to explore the oxidative stress, D, and D. The toxic mechanism of NA damage and DNA methylation in CS2 induced embryo implantation disorders.
research objective
The animal model of embryo implantation disorder caused by CS2 was not exposed at the time of implantation, and the oxidative stress, DNA damage of endometrium cells, 8-OH-dG and DNMT expression level of endometrium cells were detected at different time points and different observation points, and the oxidative stress, DNA damage and DNA methylation were analyzed in the embryo implantation barrier caused by CS2. To further deepen the understanding of the mechanism of embryo implantation barrier toxicity induced by CS2 exposure during peri implantation, and to provide a useful theoretical basis and research methods for the study of reproductive damage in environmental factors.
research method
1. research design
The research is divided into three parts. The first part is used to establish the time dependent animal model of CS2 exposure induced implantation barrier. Through the embryo implantation obstacle phenomenon, it searches for the time point of CS2 induced embryo implantation barrier sensitive exposure time, and detects the level of oxidative stress in the uterus tissue of pregnant mice, and analyzes the role of oxidative stress in the mechanism of CS2 induced embryo implantation barrier. In the second part, on the basis of the first part of the experiment, the pregnant rats were exposed to CS2 at the time of CS2 induced implantation barrier, and the DNA injury and the 8-OH-dG level of endometrium in the endometrium of pregnant rats were detected at different point of time. The effect of DNA damage and the oxidative damage and oxidation of DNA in the mechanism of CS2 induced embryo implantation barrier toxicity and DNA damage and oxidation were analyzed. The relationship between stress. The third part of the animals treated the same second parts to detect the expression level of DNMT in the uterus tissue of pregnant mice, and analyze the role of DNA methylation in the mechanism of CS2 induced embryo implantation barrier toxicity.
2. exposure time
This experiment was divided into 4 treatments according to the different exposure time of C82, that is, the animals were treated with a treatment (C82 or solvent) at pregnant 3D, pregnant 4D, pregnant 5D and pregnant 6D (GD3, GD4, GD5 and GD6).
3. the end point of observation
The continuous time points after exposure were selected as the observation end point, and there were 26 observation points in the four treatment. The first treatment group had 8 observation endpoints at 6h, 12h, 18h, 24h, 48h, 72h, 96h and 144H (GD9) respectively after exposure, and the second treatment groups had 7 observation endpoints after exposure. The third treatment groups had 6 observation points for 6h, 12h, 18h, 24h, 48h and 96h (GD9) after exposure, and the fourth treatment groups had 5 observation points in 6h, 12h, 18h, 24h and 72h (i.e., 72h) after exposure.
The final 1 end points of the treatment group (GD9) were used to observe maternal toxicity and the number of embryo implantation. The other observation endpoints were used to detect oxidative stress, DNA damage and DNMT1 expression.
Each observation endpoint contained 1 CS2 exposure groups and 1 solvent control groups.
4. animal conception and grouping
Before the experiment, the clean grade mature Kunming mice were fed for one week, according to the female and male 1:1 cage, the next day morning examination of the Yin suppository, the positive suppositories were recorded as the first day of pregnancy (GD1), and were randomly divided into 26 observation points. Each observation end group contained 12 pregnant rats, and then randomly assigned to the exposed and control groups (each containing 6 pregnant mice).
Treatment of 5. pregnant rats
The pregnant rats received 1 intraperitoneal injection (CS2 exposure dose of 631.4mg/kg, pre collocation, and the control group of olive oil), respectively, according to the exposure time point of experimental design, and the volume of injection was 0.1m1/10g weight.
6. sample collection
The pregnant rats at the last point of observation (GD9) of the 4 treatment groups were killed in the dislocated 9D of pregnancy, and the uterus, ovary, liver, spleen, kidney and embryo were taken and weighed, and the number of embryo implantation was counted. The pregnant rats in the 4 treatment groups were taken out of the uterus after the dislocated end of the design, and half of the uterus tissue was removed by manual homogenate. The rats in the other half of the uterus were scraped from the endometrial cells of the pregnant rats by manual mechanical scraping for DNA damage detection. -80
7. the expression of oxidative stress, DNA damage, 8-OH-dG and DNMT in uterus of pregnant rats
Using the method of Coomassie brilliant blue to quantify the protein of the uterus tissue homogenate of the exposed and control groups, the levels of malondialdehyde (MDA), peroxidase (SOD), glutathione peroxidase (GSH-PX) and peroxisase (CAT) in the uterine tissue homogenate of pregnant mice were measured by spectrophotometry, and the enzyme linked immunosorbent assay (enz) was used. Ymes linked immunosorbent assay, ELISA) determination of 8-OH-dG content in uterus tissue of pregnant mice; detection of DNA damage in endometrium cells of pregnant rats by comet assay (comet assay); Determination of uterus group by twelve sodium alkyl sulfonate polyacrylamide gel electrophoresis (SDS-PAGE) and protein immunoblotting (Western blotting) method The content of methyltransferase 1 (DNAmethyltransferase1, DNMT1) in weaving.
8. statistical analysis
SPSS20.0 was used to carry out statistical analysis. After normality test, the measurement indexes of normal and approximate normal distribution were all expressed in X + s. First, each index was tested for variance homogeneity: Kamo Sai, F test was carried out by single factor analysis of variance (ONE-WAY ANOVA), and Dunnett's t test was used to compare the exposure group and the control group. If the variance is not homogeneous, the non parametric statistical method (Kruskal-Wallis H method) is adopted and the correlation analysis is used to analyze the relationship between the indexes. The statistical test level is set as alpha =0.05.
Research results
1. the toxicity of exposure to CS2 on maternal and embryo in pregnant rats.
Maternal toxicity: after exposure to CS2 at different time points of embryo implantation in pregnant mice, there was no significant difference between the exposed groups and the control group (P0.05), and there was no significant difference between the pregnant rats' uterus, the ovary, the liver, the spleen and the kidney (P0.05). The difference was not statistically significant (P0.05). The maternal toxicity caused by the quantity is small.
Embryo toxicity: the number of embryo implantation in each exposure group was significantly lower than that of the control group (P0.01).GD3.GD4. GD5 and GD6, the embryo implantation rates were 56.95%, 36.26%, 39.55% and 52.74% respectively, compared with the control group, the difference was statistically significant (P0.01), the embryo implantation rate after exposure to CS2 was significantly lower, and the embryo implantation rate in GD4 exposure group was the lowest (P0.05). There was no significant difference in embryo weight after correction of embryo implantation (P0.05).
2. the effect of CS2 exposure on oxidative stress in the uterus of pregnant rats during implantation period
After the exposure of CS2, the level of MDA in the uterus tissues of the exposed rats was significantly higher than that in the control group (P0.01). The MDA level of the uterus tissue in the GD4 exposed group was 131.4% higher than that in the control group. GD3, GD5 and GD6 were increased by 120.8%, 121.6% and 104.9%. respectively, respectively, and the different observation points of the different exposure points were observed at different points of exposure. The levels of SOD, GSH-PX and CAT in the uterus tissue of pregnant rats were significantly lower than those in the control group (P0.01): CAT level decreased significantly (P0.01) after exposure (P0.01), GSH-PX level was significantly reduced after exposure (P0.01) and SOD levels decreased after exposure. The correlation analysis showed that the uterus was exposed to the uterus after exposure. There was a significant negative correlation between MDA level and GD9 embryo implantation number (r=-0.783, P0.01).
3. the effect of CS2 exposure on DNA damage in endometrium of pregnant rats
The indexes of DNA injury in endometrium cells of pregnant rats (TL, TM, OTM and TDNA%) increased significantly after CS2 exposure (P0.01), and reached the highest point at 18h after exposure (P0.01). Negative correlation (r=-0.804, -0.847, -0.934 and -0.863, P0.01).
4. the effect of CS2 exposure on 8-OH-dG level in the uterus of pregnant rats
Compared with the control group, the 8-OH-dG level of uterus tissue in pregnant rats was significantly increased at 18h and 24h after CS2 exposure. The difference was statistically significant (P0.01). The level of 8-OH-dG in uterus tissue of pregnant rats was 893.8% and 647.4% (P0.01) compared with the control group at 18h and 24h.
5. the effect of CS2 exposure on DNMT1 level in the uterus of pregnant rats
The level of DNMT1 expression in uterus tissue of pregnant rats decreased significantly at 6h after GD4 exposure to CS2, and the level of DNMT1 expression in uterus tissue of pregnant rats increased significantly at 12h after exposure. The difference was statistically significant (P0.01) compared with the control group (P0.01). The correlation analysis showed that the expression level of DNMT1 and the number of embryo implantation in the uterine tissue of 12h pregnant rats after CS2 exposure were compared. There was a significant negative correlation between them (r=-0.433, P0.01).
6. the relationship between oxidative stress, DNA damage and 8-OH-dG level and CS2 exposure
There was a significant change in the DNA damage index 6h after CS2 exposure to GD4 in pregnant rats, and the increase of MDA in the uterus tissue was statistically significant when exposed to CS2 after 18h, suggesting that the DNA damage in the uterus tissue of pregnant mice induced by CS2 was earlier than the change of oxidative stress. The results of the correlation analysis showed that 8-OH-dG and DNA damage indicators were used as the biomarkers of DNA oxidative damage. There was a positive correlation between DNA%, TL, TM and OTM (r=0.766,0.688,0.738 and 0.771, P0.01), and the degree of DNA damage to the endometrium cells reached a peak at the highest point of oxidative stress, suggesting that oxidative stress caused by oxidative stress further aggravated the degree of DNA damage in the uterus tissue of pregnant mice.
conclusion
The 1. peri implantation period was exposed to C at different time
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R714.2

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