酒精对小鼠生殖细胞及植入前胚胎表观遗传修饰的影响

发布时间：2019-03-20 14:49

【摘要】：研究表明酒精可导致后代出现一些典型的生理缺欠,如患者的面部会出现扭曲,中枢神经系统发育不完善,等他们成年后语言表达障碍,记忆能力明显的下降及缺乏人际沟通能力,这在医学上叫做胎儿酒精综合征(Fetal Alcohol Spectrum Disorders, FASD)。该病关于表观遗传修饰的发病机制成为研究热点,而国内外主要集中在对神经系统发育水平的研究。众所周知植入前胚胎有序的表观遗传模式对后期胚胎的发育异常的重要,若出现了紊乱,则会导致早期胎儿发育异常及成年疾病的出现。我们课题组前期工作发现,小鼠植入前胚胎的表观遗传修饰易受到环境因素影响,环境的变化可使表观遗传模式发生紊乱,于是推测酒精可使早期胚胎表观遗传修饰发生异常。表观遗传修饰主要包括DNA甲基化、组蛋白修饰及非编码RNA等,一般认为它调控有关细胞命运及胚胎发育的基因表达。DNA甲基化作为其中一种饰研究较为清楚的表观遗传修方式,酒精可干扰蛋氨酸合成与代谢,进而可影响甲基供体的正常供给,并且哺乳动物的组织因酒精摄入而导致DNA甲基化降低,故我们猜测酒精影响植入前胚胎的DNA甲基化的水平。而组蛋白氨基末端的修饰作为另一个重要的表观遗传机制,对染色质的结构进行调节而调控基因的表达,例如,组蛋白的甲基化与乙酰化在基因表达调控中起着重要作用,并影响生物体的基本发育进程。在父性、母性孕期酒精暴露研究中,怀孕期间酒精摄入对植入前胚胎DNA甲基化及组蛋白修饰可能造成损害的差异,也为我们预防酒精对胎儿的伤害提供借鉴的作用。小鼠通常作为研究该病的动物模型,为了研究FASD的表观遗传的发病机制及为治疗该病提供一定的实验依据。本研究采用活体灌胃法、HE染色法、精子的质量分析技术、体内冲胚技术、胚胎体外培养法及免疫荧光技术,分析酒精对肝脏及精液质量、生殖细胞及体内、外早期胚胎的表观遗传修饰的影响。我们分别对雄鼠、雌鼠受孕前后及双亲进行灌酒处理,研究酒精对生殖细胞及植入前胚胎全基因组DNA甲基化水平的影响。后期我们进一步研究雌鼠受孕前后酒精摄入对H3K27三甲基化／乙酰化模式的影响。实验数据显示,灌酒小鼠的生殖细胞及植入前胚胎表观遗传修饰模式发生异常,具体如下：雄鼠灌酒实验表明酒精分别肝脏和精液的质量的损害,使精子的DNA甲基化水平降低,导致后期植入前胚胎DNA甲基化水平异常。受孕前后雌鼠灌酒实验结果表明酒精降低卵子及植入前胚胎DNA甲基化水平。双亲灌酒实验结果表明酒精对植入前胚胎的DNA甲基化损害程度依次为双亲灌酒雌性灌酒雄性灌酒。雌鼠受孕前后酒精摄入对植入前胚胎的H3K27三甲基化影响较不明显,而酒精对该时期胚胎的H3K27乙酰化影响较明显。酒精摄入对小鼠的生殖细胞及植入前胚胎表观遗传的的正确建立造成紊乱,为揭示胎儿酒精综合征发病机制提供重要的借鉴。为深入理解孕期酒精暴露的危害,揭示受孕酒精暴露对后代长时期的影响提供一定的实验依据,同时提供预防受孕酒精暴露对子代的伤害提供一种思路。
[Abstract]:The study shows that alcohol can cause some typical physiological defects in the offspring, such as the distortion of the face of the patient, the imperfect central nervous system, the expression of the disorder in the adult post-adult language, the obvious decline of the memory ability and the lack of interpersonal communication ability, This is medically known as the Fetus Alcohol Spectrum Disdders (FASD). The pathogenesis of epigenetic modification has become the focus of the study, and the research on the development level of the nervous system is mainly concentrated at home and abroad. It is well known that the apparent genetic pattern of pre-implantation embryo order is important to the development of later-stage embryos, and if a disorder occurs, the early fetal development and the appearance of adult diseases can be caused. The preliminary work of our research group found that the epigenetic modification of the pre-implantation embryos of the mice is susceptible to environmental factors, and the change of the environment can cause the apparent genetic pattern to be disordered, so that the apparent genetic modification of the early embryo may be abnormal. Epigenetic modification mainly includes DNA methylation, histone modification and non-coding RNA. It is generally considered that it regulates the genes related to cell fate and embryonic development. D. DNA methylation as one of the more clear epigenetic methods in which alcohol can interfere with the synthesis and metabolism of methionine, which in turn affects the normal supply of methyl donor, and the tissue of a mammal results in a DNA methylation down due to alcohol intake. Low, so we suspect that alcohol affects the DNA methylation of the pre-implantation embryos The modification of the amino-terminal of histone is another important epigenetic mechanism, and the structure of chromatin is regulated to regulate the expression of the gene. For example, the methylation of histone and ethylation of histone play an important role in the regulation of gene expression and affect the basic development of the organism. Cheng. During the study of maternal and maternal alcohol exposure, alcohol intake during pregnancy can cause damage to pre-implantation fetal DNA methylation and histone modification, and also provide reference for our prevention of alcohol damage to the fetus. The mouse is usually used as an animal model for the study of the disease. In order to study the pathogenesis of the apparent inheritance of the FASD and to provide some real evidence for the treatment of the disease In this study, the epigenetic modification of the quality of the liver and the semen, the germ cells and the in vivo and in vitro embryos of the liver and the semen, the germ cells and the in vivo and in vitro were analyzed by the method of living in-life, HE staining, the quality analysis of the sperm, the in-vivo embryo-flushing technique, the in vitro culture of the embryo and the immunofluorescence technique. The effects of alcohol on the DNA methylation of the whole genome of the germ cells and the pre-implantation embryos were studied. The effect of alcohol intake on H3K27 trimethylation/ ethylation patterns before and after conception in female rats was further studied in the later stage. The experimental data showed that the germ cells and the epigenetic modification of the pre-implantation embryos of the mice were abnormal, as follows: The experimental data of the male rats showed that the quality of the liver and the semen of the alcohol was damaged, so that the DNA of the sperm was methylated. reduced level, resulting in post-implantation of pre-implantation embryo DNA methylation water The results of the experiment of the female rats before and after conception showed that the alcohol decreased the egg and the pre-implantation embryo DNA methyl group. The results of the experiment of both parents and parents showed that the degree of DNA methylation of alcohol in the pre-implantation embryos was both parent and female. The effect of alcohol intake before and after the conception of female rats on the three-methylation of H3K27 in the pre-implantation embryos was not significant, and the effect of alcohol on the development of H3K27 in the embryo during the period was not significant. Obvious. The correct establishment of the apparent genetic inheritance of the germ cells and pre-implantation embryos of the mice caused by alcohol intake is a disorder, which is provided for the purpose of revealing the pathogenesis of the fetal alcohol syndrome It is an important reference. In order to understand the harm of alcohol exposure during pregnancy, it is revealed that the exposure of the pregnant alcohol to the long period of the offspring provides a certain experimental basis, and also provides the prevention of the exposure of the pregnant alcohol to the offspring.
【学位授予单位】：安徽大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R363

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