活性氧、FOXO3a与高浓度葡萄糖环下小鼠早期胚胎发育阻滞的相关性研究

发布时间：2018-05-16 21:36

本文选题：FOXO3a + ROS　；参考：《郑州大学》2014年硕士论文

【摘要】：妊娠合并糖尿病的育龄期妇女容易出现胚胎丢失、早期流产等，造成这一现象的原因可能与其血浆葡萄糖浓度过高导致胚胎发育异常密切相关。研究表明，早期胚胎暴露于高浓度葡萄糖环境下,其生长发育受抑制，更容易发生胚胎发育阻滞。然而，高血糖抑制早期胚胎发育的具体机制尚不清楚。已有的研究表明，高葡萄糖环境中，活性氧簇（Reactive oxygen species,ROS）的产生量显著增加。通过ROS介导的氧化应激损伤，细胞内的脂质、蛋白质发生过氧化，DNA链断裂，严重时即可引起胚胎发育障碍。 FOXO3a是同ROS相联系的重要转录因子，也是目前研究细胞氧化应激损伤最重要的蛋白之一。研究表明，高浓度的葡萄糖通过诱导ROS过量的产生，导致FOXO3a被磷酸化并失去活性，并使其靶基因—FOXO3a依赖的锰过氧化物歧化酶（manganese superoxide dismutase）表达降低，进而产生更多的ROS，并加速氧化应激相关疾病的病理进程。研究显示，FOXO3a对细胞内氧化应激水平的调节作用可改善胚胎的发育，然而，FOXO3a是否参与了高浓度葡萄糖诱导氧化应激损伤而导致胚胎发育阻滞，尚须进一步研究。目的在小鼠胚胎培养基中添加不同浓度的葡萄糖，观察其生长和发育情况，同时在胚胎发育的不同时期对ROS含量和FOXO3a的表达进行检测，研究不同浓度葡萄糖条件下胚胎发育阻滞与ROS、FOXO3a的相关性，试图从胚胎发育的角度探讨糖尿病妇女妊娠失败的机理，并为其临床治疗提供理论依据。方法对昆明系SPF级雌性小鼠行超促排卵处理，注射HCG后合笼，断颈法处死小鼠后收集合子，基于胚胎培养基中补充葡萄糖溶液后葡萄糖终浓度的高低而将实验分为四组：control组（不添加葡萄糖）、5mM组、15mM组和25mM组，将合子分别置于以上四个分组的培养基内培养，观察胚胎在不同浓度葡萄糖条件下的发育情况。在胚胎发育的不同阶段（合子期、2-细胞期、4-细胞期及囊胚期），通过DCFH-DA染料对胚胎进行染色，以检测ROS的含量，胚胎中FOXO3a蛋白的表达情况采用细胞免疫荧光染色法检测，同时通过Real Time PCR的方法检测FOXO3a mRNA的相对表达量。结果 1.高浓度葡萄糖影响早期小鼠胚胎发育高葡萄糖下小鼠早期胚体外培养的结果表明：随着鼠胚培养基葡萄糖液浓度的上升，鼠胚的囊胚形成率下降，并且增加了2-细胞鼠胚的发育阻滞率。15mM组与25mM组的囊胚形成率（分别为50.0%、43.3%）明显低于control组和5mM组（分别为69.9%、63.6%）（P 0.05）；而15mM组和25mM组胚胎发育阻滞率（分别为37.5%、38.9%）明显高于control组和5mM组（分别为19.4%、21.2%）（P 0.05）。 2. ROS含量的检测 ROS检测结果表明：相对于合子、4-细胞和囊胚期胚胎，小鼠2-细胞期胚胎ROS的水平明显升高（P 0.05）；15mM组与25mM组2/4-细胞阶段胚胎内ROS含量明显高于对照组与5mM组（P 0.05），囊胚阶段胚胎内ROS含量随葡萄糖浓度增加而升高，但各分组间差异无统计学意义（P 0.05）。 3. FOXO3a的表达胚胎细胞免疫荧光染色的结果显示，FOXO3a蛋白在胚胎发育的各时期均有不同程度的表达，且主要定位于细胞浆中。采用Real-time PCR的方法，在RNA水平对FOXO3a的检测结果表明：在小鼠胚胎发育的各时期，，鼠胚在不同浓度葡萄糖培养条件下FOXO3a的表达与ROS的相对含量密切相关，2-细胞期胚胎FOXO3a RNA表达水平相对于其他发育时期明显升高（P 0.05）；FOXO3a的相对表达量随着胚胎继续向囊胚发育而逐渐降低。对于相同发育阶段胚胎来说，随着糖浓度的升高，2-细胞、4-细胞期胚胎FOXO3a RNA表达水平降低，其中15mM组和25mM组显著低于control组和5mM组（P 0.05）；而对于囊胚期胚胎，FOXO3a RNA表达的组间差异无统计学意义（P0.05）。结论。 1.胚胎培养液中高浓度的葡萄糖抑制早期胚胎发育，可导致胚胎发育阻滞现象增加。 2.高浓度葡萄糖可通过诱导胚胎生成过量的ROS，对胚胎产生氧化应激损伤，从而使胚胎的发育潜能降低。 3.在高浓度葡萄糖对胚胎发育造成的氧化应激损伤中，FOXO3a可能作为ROS的下游靶点，参与了ROS介导的早期胚胎发育阻滞。
[Abstract]:Women of childbearing age of pregnancy with diabetes are prone to embryo loss, early abortion and so on. The cause of this phenomenon may be closely related to the abnormal development of the embryo. The study shows that the early embryos were exposed to high concentration of glucose, and their growth and development were inhibited and embryo development was more likely to occur. Block. However, the specific mechanism for the inhibition of early embryonic development by hyperglycemia is not clear. Previous studies have shown that the production of Reactive oxygen species (ROS) is significantly increased in the high glucose environment. Through ROS mediated oxidative stress damage, the lipid, protein peroxidation in the cells, and the rupture of the DNA chain in the cells can be induced seriously. An embryonic development disorder.
FOXO3a is an important transcription factor associated with ROS, and is one of the most important proteins to study oxidative stress in cells. The study shows that the high concentration of glucose is induced by the excessive production of ROS, resulting in the phosphorylation of FOXO3a and the loss of activity, and the target gene, FOXO3a dependent manganese peroxide dismutase (manganese superoxide). Dismutase) decrease in expression, produce more ROS, and accelerate the pathological process of oxidative stress related diseases. The study shows that the regulation of FOXO3a on the level of intracellular oxidative stress can improve the development of embryos. However, whether FOXO3a is involved in the inhibition of oxidative stress induced by high concentration of glucose and the development of embryonic retardation is still necessary. Step by step.
objective
The growth and development of glucose were observed with different concentrations of glucose in the mouse embryo culture medium. At the same time, the content of ROS and the expression of FOXO3a were detected at different stages of embryo development. The correlation of embryo development block with ROS and FOXO3a under different concentrations of glucose was studied, and the diabetic women were explored from the angle of embryo development. The mechanism of pregnancy failure and provide a theoretical basis for its clinical treatment.
Method
The female mice of Kunming grade SPF were treated with Super Ovulation, HCG cages were injected, and the zygotes were collected after the neck broken method. The experiment was divided into four groups based on the glucose terminal concentration in the embryo culture medium supplemented with glucose solution. The control group (no glucose), 5mM, 15mM and 25mM groups were placed in the above four groups respectively. In the medium of culture, the development of embryos at different concentrations of glucose was observed. At different stages of embryo development (zygote stage, 2- cell stage, 4- cell phase and blastocyst stage), the embryo was stained with DCFH-DA dye to detect the content of ROS. The expression of FOXO3a protein in embryo was stained by cell immunofluorescence staining. At the same time, the relative expression of FOXO3a mRNA was detected by Real Time PCR.
Result
1. high concentration of glucose affects early mouse embryonic development
In vitro culture of early embryos in high glucose mice showed that with the increase of glucose concentration in the rat embryo culture medium, the blastocyst formation rate of mouse embryos decreased, and the formation rate of the development block rate of 2- cells.15mM group and 25mM group (50%, 43.3% respectively) was significantly lower than that of group control and 5mM group (69.9%, 63.6% respectively). P 0.05); the retardation rate of embryonic development in group 15mM and group 25mM (37.5%, 38.9%) was significantly higher than that in group control and 5mM (19.4%, 21.2%, respectively) (P 0.05).
Detection of 2. ROS content
ROS test results showed that compared with the zygote, 4- and blastocyst embryos, the level of ROS in mouse 2- cell stage embryos increased significantly (P 0.05), and ROS content in 15mM and 25mM group 2/4- cell stage embryos was significantly higher than that of control group and 5mM group (P 0.05), and the content of ROS in blastocyst stage embryos increased with the increase of glucose concentration, but the difference between the groups was poor. The difference was not statistically significant (P 0.05).
The expression of 3. FOXO3a
The results of cell immunofluorescence staining showed that FOXO3a protein was expressed in varying degrees at various stages of embryonic development and mainly located in the cytoplasm. The results of the detection of FOXO3a at the level of RNA by the method of Real-time PCR showed that the mouse embryo was FO under the conditions of different concentration of glucose at various time periods of mouse embryo development. The expression of XO3a was closely related to the relative content of ROS. The expression level of FOXO3a RNA in 2- cell phase embryos was significantly higher than that of other developmental stages (P 0.05). The relative expression of FOXO3a decreased gradually as the embryo continued to develop to the blastocyst. For the same developmental stage embryos, with the increase of sugar concentration, 2- cells, 4- cell embryos FO. The expression level of XO3a RNA was decreased, among which group 15mM and 25mM were significantly lower than group control and 5mM (P 0.05), but there was no significant difference between the groups of FOXO3a RNA expression for the blastocyst embryo (P0.05).
Conclusion.
1. high concentration of glucose in embryo culture inhibits early embryo development, which may lead to an increase in embryonic development retardation.
2. high glucose can induce oxidative stress damage to embryos by inducing excessive ROS production, thereby reducing the developmental potential of embryos.
3. in the oxidative stress damage caused by high concentration of glucose on embryonic development, FOXO3a may be a downstream target of ROS and participates in the early embryonic development block mediated by ROS.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R714.256

【参考文献】