本文選題：FOXO3a + ROS��； 參考：《鄭州大學(xué)》2014年碩士論文

【摘要】：妊娠合并糖尿病的育齡期婦女容易出現(xiàn)胚胎丟失、早期流產(chǎn)等，造成這一現(xiàn)象的原因可能與其血漿葡萄糖濃度過高導(dǎo)致胚胎發(fā)育異常密切相關(guān)。研究表明，早期胚胎暴露于高濃度葡萄糖環(huán)境下,其生長發(fā)育受抑制，更容易發(fā)生胚胎發(fā)育阻滯。然而，高血糖抑制早期胚胎發(fā)育的具體機制尚不清楚。已有的研究表明，高葡萄糖環(huán)境中，活性氧簇（Reactive oxygen species,ROS）的產(chǎn)生量顯著增加。通過ROS介導(dǎo)的氧化應(yīng)激損傷，細胞內(nèi)的脂質(zhì)、蛋白質(zhì)發(fā)生過氧化，DNA鏈斷裂，嚴(yán)重時即可引起胚胎發(fā)育障礙。 FOXO3a是同ROS相聯(lián)系的重要轉(zhuǎn)錄因子，也是目前研究細胞氧化應(yīng)激損傷最重要的蛋白之一。研究表明，高濃度的葡萄糖通過誘導(dǎo)ROS過量的產(chǎn)生，導(dǎo)致FOXO3a被磷酸化并失去活性，并使其靶基因—FOXO3a依賴的錳過氧化物歧化酶（manganese superoxide dismutase）表達降低，進而產(chǎn)生更多的ROS，并加速氧化應(yīng)激相關(guān)疾病的病理進程。研究顯示，F(xiàn)OXO3a對細胞內(nèi)氧化應(yīng)激水平的調(diào)節(jié)作用可改善胚胎的發(fā)育，然而，F(xiàn)OXO3a是否參與了高濃度葡萄糖誘導(dǎo)氧化應(yīng)激損傷而導(dǎo)致胚胎發(fā)育阻滯，尚須進一步研究。 目的 在小鼠胚胎培養(yǎng)基中添加不同濃度的葡萄糖，觀察其生長和發(fā)育情況，同時在胚胎發(fā)育的不同時期對ROS含量和FOXO3a的表達進行檢測，研究不同濃度葡萄糖條件下胚胎發(fā)育阻滯與ROS、FOXO3a的相關(guān)性，試圖從胚胎發(fā)育的角度探討糖尿病婦女妊娠失敗的機理，并為其臨床治療提供理論依據(jù)。 方法 對昆明系SPF級雌性小鼠行超促排卵處理，注射HCG后合籠，斷頸法處死小鼠后收集合子，基于胚胎培養(yǎng)基中補充葡萄糖溶液后葡萄糖終濃度的高低而將實驗分為四組：control組（不添加葡萄糖）、5mM組、15mM組和25mM組，將合子分別置于以上四個分組的培養(yǎng)基內(nèi)培養(yǎng)，觀察胚胎在不同濃度葡萄糖條件下的發(fā)育情況。在胚胎發(fā)育的不同階段（合子期、2-細胞期、4-細胞期及囊胚期），通過DCFH-DA染料對胚胎進行染色，以檢測ROS的含量，胚胎中FOXO3a蛋白的表達情況采用細胞免疫熒光染色法檢測，同時通過Real Time PCR的方法檢測FOXO3a mRNA的相對表達量。 結(jié)果 1.高濃度葡萄糖影響早期小鼠胚胎發(fā)育 高葡萄糖下小鼠早期胚體外培養(yǎng)的結(jié)果表明：隨著鼠胚培養(yǎng)基葡萄糖液濃度的上升，鼠胚的囊胚形成率下降，并且增加了2-細胞鼠胚的發(fā)育阻滯率。15mM組與25mM組的囊胚形成率（分別為50.0%、43.3%）明顯低于control組和5mM組（分別為69.9%、63.6%）（P 0.05）；而15mM組和25mM組胚胎發(fā)育阻滯率（分別為37.5%、38.9%）明顯高于control組和5mM組（分別為19.4%、21.2%）（P 0.05）。 2. ROS含量的檢測 ROS檢測結(jié)果表明：相對于合子、4-細胞和囊胚期胚胎，小鼠2-細胞期胚胎ROS的水平明顯升高（P 0.05）；15mM組與25mM組2/4-細胞階段胚胎內(nèi)ROS含量明顯高于對照組與5mM組（P 0.05），囊胚階段胚胎內(nèi)ROS含量隨葡萄糖濃度增加而升高，但各分組間差異無統(tǒng)計學(xué)意義（P 0.05）。 3. FOXO3a的表達 胚胎細胞免疫熒光染色的結(jié)果顯示，F(xiàn)OXO3a蛋白在胚胎發(fā)育的各時期均有不同程度的表達，且主要定位于細胞漿中。采用Real-time PCR的方法，在RNA水平對FOXO3a的檢測結(jié)果表明：在小鼠胚胎發(fā)育的各時期，，鼠胚在不同濃度葡萄糖培養(yǎng)條件下FOXO3a的表達與ROS的相對含量密切相關(guān)，2-細胞期胚胎FOXO3a RNA表達水平相對于其他發(fā)育時期明顯升高（P 0.05）；FOXO3a的相對表達量隨著胚胎繼續(xù)向囊胚發(fā)育而逐漸降低。對于相同發(fā)育階段胚胎來說，隨著糖濃度的升高，2-細胞、4-細胞期胚胎FOXO3a RNA表達水平降低，其中15mM組和25mM組顯著低于control組和5mM組（P 0.05）；而對于囊胚期胚胎，F(xiàn)OXO3a RNA表達的組間差異無統(tǒng)計學(xué)意義（P0.05）。 結(jié)論。 1.胚胎培養(yǎng)液中高濃度的葡萄糖抑制早期胚胎發(fā)育，可導(dǎo)致胚胎發(fā)育阻滯現(xiàn)象增加。 2.高濃度葡萄糖可通過誘導(dǎo)胚胎生成過量的ROS，對胚胎產(chǎn)生氧化應(yīng)激損傷，從而使胚胎的發(fā)育潛能降低。 3.在高濃度葡萄糖對胚胎發(fā)育造成的氧化應(yīng)激損傷中，F(xiàn)OXO3a可能作為ROS的下游靶點，參與了ROS介導(dǎo)的早期胚胎發(fā)育阻滯。
[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.
【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R714.256

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相關(guān)期刊論文 前2條

1 張雷;李鐘淑;方南洙;;葡萄糖在小鼠胚胎2-細胞晚期誘導(dǎo)活性氧增多抑制胚胎體外發(fā)育[J];第二軍醫(yī)大學(xué)學(xué)報;2009年08期

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