植入期二硫化碳暴露对小鼠子宫组织氧化应激、DNA损伤及甲基转移酶表达的影响

发布时间：2018-06-09 04:50

本文选题：二硫化碳 + 氧化应激　；参考：《山东大学》2014年硕士论文

【摘要】：研究背景二硫化碳(Carbon disulfide, CS2)是工业上常用的有机溶剂和化工原料,广泛用于粘胶纤维、玻璃纸、橡胶等的生产过程中。CS2具有多系统毒性,长期低剂量暴露于CS2可导致神经、心血管、内分泌系统等的病变。此外,值得关注的是CS2具有明显的生殖损伤毒作用,可导致男性睾丸萎缩、精子质量下降、性功能减退、性激素分泌紊乱等,并可导致女性月经周期紊乱、自然流产、早产等。本课题组通过前瞻性队列研究发现,暴露组女工早早孕丢失率显著高于对照组,并且暴露组女工的妊娠时间与对照组相比明显延长,说明CS2影响胚胎的着床过程。动物实验研究发现,小鼠在围植入期暴露于CS2导致胚胎植入数目明显减少,但其毒性作用机制尚未明确。氧化应激可以导致脂质过氧化、DNA损伤及蛋白质结构和功能的改变。研究表明,接触CS2可引起机体氧化应激反应,机体处于高氧化应激水平可增加自然流产的风险。提示孕期CS2暴露可以通过改变机体的氧化应激水平而影响胚胎正常着床。课题组前期实验结果显示,胚胎植入期暴露于CS2可导致孕鼠子宫内膜细胞DNA损伤,但与氧化应激的关系尚不明确。DNA甲基化可以影响基因转录过程调节植入相关蛋白的表达水平,影响胚胎植入。DNA甲基化在调节基因功能过程中需要甲基转移酶(DNA methyltransferase, DNMT)的参与。Logan等研究发现,低甲基化水平可以增强子宫内膜对胚胎的容受性,有利于胚胎植入。氧化应激可引起机体DNA甲基化水平的改变,同时氧化应激导致的DNA氧化损伤可启动机体的碱基切除修复过程,而参与碱基切除修复的有关酶需要经过甲基化后才能发挥作用。综上,我们推测在DNA氧化损伤后,需要DNMT的高表达以启动DNA损伤修复过程。因此,我们假设在孕鼠暴露于CS2后,氧化应激、DNA损伤和DNA甲基化的单独或联合作用是胚胎植入障碍的重要机制。本研究拟运用不同胚胎植入时段CS2暴露与胚胎植入障碍关系的时间依赖动物模型,检测暴露后不同染毒时间点和观察终点孕鼠子宫组织的氧化应激、子宫内膜细胞DNA损伤、DNA氧化损伤生物标志物8-OH-dG以及DNMT表达水平,探讨氧化应激、DNA损伤及DNA甲基化在CS2致胚胎植入障碍中的毒性作用机制。研究目的构建胚胎植入期不同时点暴露于CS2致胚胎植入障碍动物模型,检测不同染毒时间点与不同观察终点的孕鼠子宫组织氧化应激、子宫内膜细胞DNA损伤、8-OH-dG以及DNMT表达水平,分析氧化应激、DNA损伤和DNA甲基化在CS2所致的胚胎植入障碍中的作用及其致病机制,进一步加深对围植入期CS2暴露所致胚胎植入障碍毒作用机制的了解,为环境因素生殖损伤研究提供可借鉴的理论依据和研究方法。研究方法 1.研究设计研究设计分为三部分。第一部分用于建立CS2暴露致胚胎植入障碍时间依赖动物模型,通过胚胎植入障碍现象寻找CS2致胚胎植入障碍敏感暴露时间点,并检测孕鼠子宫组织氧化应激水平,分析氧化应激在CS2致胚胎植入障碍机制中的作用。第二部分在第一部分实验的基础上,选择CS2致胚胎植入障碍敏感时点使孕鼠暴露于CS2,并在不同时点终止实验,检测孕鼠子宫内膜细胞DNA损伤和子宫组织8-OH-dG水平,分析在CS2致胚胎植入障碍毒作用机制中DNA损伤的作用及DNA损伤与氧化应激的关系。第三部分动物处理同第二部分,检测孕鼠子宫组织中DNMT表达水平,分析在CS2致胚胎植入障碍毒作用机制中DNA甲基化的作用。 2.暴露时间本实验根据C82暴露时间的不同分为4种处理,即动物分别在孕3d、孕4d、孕5d和孕6d(记为GD3、GD4、GD5和GD6)接受一次处理(C82或溶剂)。 3.观察终点选取暴露后的连续时间点作为观察终点,四种处理共有26个观察终点,即第一个处理组分别在暴露后第6h、12h、18h、24h、48h、72h、96h和144h(即GD9)共设有8个观察终点；第二个处理组分别在暴露后6h、12h、18h、24h、48h、72h和120h(即GD9)共设有7个观察终点；第三个处理组分别在暴露后6h、12h、18h、24h、48h和96h(即GD9)共设有6个观察终点；第四个处理组分别在暴露后6h、12h、18h、24h和72h(即GD9)共设有5个观察终点。各处理组的最后1个观察终点(GD9)用于观察母体毒性及胚胎植入数目,其余各观察终点用于检测氧化应激、DNA损伤及DNMT1表达水平等。每个观察终点包含1个CS2暴露组和1个溶剂对照组。 4.动物受孕与分组实验前清洁级性成熟昆明种小鼠适应性喂养一周,按雌雄1：1合笼,次日晨检查阴栓,阴栓阳性者记为孕第一天(记为GD1),随机分到26个观察终点。每个观察终点组含12只孕鼠,再随机分配进入暴露组和对照组(各含6只孕鼠)。 5.孕鼠处理孕鼠分别按照实验设计的暴露时点接受1次腹腔注射(CS2暴露剂量为631.4mg/kg,染毒前配置；对照组为橄榄油)；注射容量为0.1m1/10g体重。 6.样本收集 4种处理组中最后一个观察终点(GD9)的孕鼠在孕9d经脱臼处死,取子宫、卵巢、肝、脾、肾和胚胎并称重,计数胚胎植入数目；4种处理组中其他观察终点的孕鼠在设计的观察终点经脱臼处死后取子宫组织,一半子宫组织经手动匀浆后取上清液,-80℃冰箱保存备用。另一半子宫组织采用手工机械刮取法直接刮取孕鼠子宫内膜细胞用于DNA损伤测定。 7.孕鼠子宫组织氧化应激、DNA损伤、8-OH-dG和DNMT表达水平的测定运用考马斯亮蓝法对暴露组和对照组孕鼠子宫组织匀浆液进行蛋白定量；运用分光光度法测定孕鼠子宫组织匀浆液中丙二醛(MDA)、过氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-PX)和过氧化氢酶(CAT)水平；运用酶联免疫吸附测定法(enzymes linked immunosorbent assay, ELISA)测定孕鼠子宫组织中8-OH-dG含量；应用彗星实验(comet assay)检测孕鼠子宫内膜细胞DNA损伤水平；运用十二烷基磺酸钠-聚丙烯酰胺凝胶电泳技术(SDS-PAGE)和蛋白免疫印迹法(western blotting)法测定孕鼠子宫组织中甲基转移酶1(DNAmethyltransferase1, DNMT1)含量。 8.统计学分析使用SPSS20.0进行统计学分析,经正态性检验,正态及近似正态分布的测量指标均以x±s表示。首先对各指标进行方差齐性检验：若方差齐,则采用单因素方差分析(ONE-WAY ANOVA)进行F检验,同时采用Dunnett's t test进行暴露组和对照组的比较；若方差不齐,则采用非参数统计方法(Kruskal-Wallis H法)。并运用相关分析分析各指标间的关系。统计学检验水准设定为α=0.05。研究结果 1.围植入期暴露于CS2对孕鼠母体及胚胎的毒性作用母体毒性：孕鼠在胚胎植入不同时间点暴露于CS2后,孕鼠GD9体重和体重净增量各暴露组与对照组比较,差异无统计学意义(P0.05)；孕鼠子宫、卵巢、肝、脾以及肾等脏器的重量及脏器系数组间比较,差异无统计学意义(P0.05)。说明该暴露剂量所致的母体毒性较小。胚胎毒性：各暴露组胚胎植入数目均明显低于对照组(P0.01)。GD3.GD4. GD5和GD6各暴露组的胚胎植入率分别为56.95%、36.26%、39.55%和52.74%,与对照组比较差异均有统计学意义(P0.01),暴露CS2后胚胎植入率明显降低,GD4暴露组的胚胎植入率最低(P0.05)。在校正胚胎植入数目后胚胎均重与对照组比较,差异无统计学意义(P0.05)。 2.围植入期暴露于CS2对孕鼠子宫组织氧化应激的影响 CS2暴露后18h,各暴露组孕鼠子宫组织中MDA水平明显升高,与对照组比较差异有统计学意义(P0.01),其中GD4暴露组孕鼠子宫组织MDA水平较对照组增高131.4%, GD3、GD5和GD6较对照组分别增高120.8%、121.6%和104.9%。此外,不同暴露时间点不同观察终点孕鼠子宫组织中SOD、GSH-PX和CAT水平明显降低,与对照组比较差异有统计学意义(P0.01):CAT水平在暴露后12h明显降低(P0.01),GSH-PX水平在暴露后12h和18h明显降低(P0.01), SOD水平在暴露后18h显著降低(P0.01)。相关分析结果显示,暴露后18h孕鼠子宫组织中MDA水平和GD9胚胎植入数目呈明显负相关关系(r=-0.783,P0.01)。 3.围植入期暴露于CS2对孕鼠子宫内膜细胞DNA损伤的影响孕鼠子宫内膜细胞DNA损伤各项指标(TL、TM、OTM和TDNA%)在CS2暴露后6h时明显升高(P0.01),并在暴露后18h时达到最高点(P0.01),之后各指标值逐渐下降。相关分析结果显示,CS2暴露后18h时孕鼠子宫内膜细胞TM、OTM、TL和TDNA%与胚胎植入数目之间呈明显负相关关系(r=-0.804、-0.847、-0.934和-0.863,P0.01)。 4.围植入期暴露于CS2对孕鼠子宫组织8-OH-dG水平的影响与对照组比较,孕鼠CS2暴露后18h和24h时孕鼠子宫组织8-OH-dG水平显著增高,差异具有统计学意义(P0.01)。在18h和24h时孕鼠子宫组织8-OH-dG水平与对照组比较,分别升高893.8%和647.4%(P0.01)。 5.围植入期暴露于CS2对孕鼠子宫组织DNMT1水平的影响孕鼠在GD4暴露于CS2后6h时孕鼠子宫组织DNMT1表达水平明显降低,而在暴露后12h时孕鼠子宫组织DNMT1表达水平明显升高,与对照组比较差异均有统计学意义(P0.01)。相关分析结果显示,CS2暴露后12h孕鼠子宫组织DNMT1表达水平与胚胎植入数目之间呈明显负相关关系(r=-0.433,P0.01)。 6.氧化应激、DNA损伤和8-OH-dG水平与CS2暴露的关系孕鼠GD4暴露于CS2后DNA损伤各项指标6h时出现明显改变,而暴露于CS2后18h时子宫组织MDA升高具有统计学意义,提示CS2所致孕鼠子宫组织DNA损伤改变早于氧化应激变化。相关分析结果显示,作为DNA氧化损伤生物标志物的8-OH-dG与DNA损伤指标TDNA%、TL、TM和OTM之间呈正相关(r=0.766、0.688、0.738和0.771,P0.01),且在氧化应激最高点时子宫内膜细胞DNA损伤程度达到高峰,提示氧化应激引起的DNA氧化损伤进一步加重孕鼠子宫组织的DNA损伤程度。结论 1.围植入期不同时间暴露于CS2均可导致孕鼠子宫组织发生明显且短暂的氧化应激反应,呈现出明显的时间变化规律且变化趋势一致,与暴露时点无关。 2.围植入期暴露于C82可导致孕鼠子宫内膜细胞DNA损伤,并呈现出明显的时间变化规律；氧化应激通过引起DNA氧化损伤进一步加重子宫内膜细胞的DNA损伤程度。 3.子宫组织氧化应激水平和内膜细胞DNA损伤水平与CS2致胚胎植入障碍程度一致,两者协同作用可能是CS2暴露致胚胎植入障碍的重要机制之一。 4.围植入期暴露于CS2导致的子宫组织DNMT1表达水平的变化,可能与机体在氧化应激和细胞DNA损伤毒性作用条件下对子宫内膜容受性的调节机制有关。
[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
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R714.2

【参考文献】