褪黑素缓解老化卵母细胞体外受精后发育受阻的研究

发布时间：2018-03-19 00:20

本文选题：卵母细胞老化　切入点：H_2O_2　出处：《中国人民解放军军事医学科学院》2017年硕士论文　论文类型：学位论文

【摘要】：高龄妇女接受体外受精(in vitro fertilization,IVF)治疗后的成功率明显偏低。其中,卵母细胞老化造成的胚胎发育能力下降,是导致高龄妇女体外受精后妊娠率低、流产率高的重要原因。因此,寻找能够缓解老化卵母细胞IVF后发育受阻的方法具有重要的意义。本实验以过氧化氢(H_2O_2)处理小鼠卵母细胞建立的氧化应激诱导老化模型为研究对象,研究氧化应激诱导卵母细胞老化后线粒体的相关特征受损与老化卵母细胞IVF后胚胎发育率及质量下降的关系。继而,以老化卵母细胞囊胚发育率和囊胚质量为主要评价指标,并辅于线粒体功能状态,考察褪黑素改善老化卵母细胞IVF胚胎发育率及质量的可行性,为提高临床高龄女性IVF成功率提供实验依据。本研究分别利用10μM、50μM、100μM和150μM不同浓度的H_2O_2处理MII期卵母细胞3 h,建立以氧化应激为特征的卵母细胞老化模型,以未经H_2O_2处理的卵母细胞为对照。各组卵母细胞进行标准的IVF后,观察发育效率。结果显示,随H_2O_2浓度的增加,卵母细胞中的氧化应激程度是明显加剧的。经150μM H_2O_2处理的卵母细胞IVF后,碎裂率相比对照组显著性升高(7.77±4.78%vs.39.32±9.14%,P0.05);100μM H_2O_2处理后2细胞率相比对照组显著降低(45.63±2.6%vs.54.67±8.27%,P0.05);当IVF胚胎发育至囊胚阶段时,50μM、100μM处理组囊胚率分别为26.27±0.06%、28.46±3.45%,均显著低于对照组(34.9±1.77%,P0.05)。这说明老化卵母细胞中的氧化应激,可能会对IVF后胚胎产生持续的影响,造成发育损伤。在许多细胞类型中,老化往往伴随着明显的线粒体功能损伤,因此我们对H_2O_2诱导卵母细胞老化的线粒体功能进行了专门的检测。活性线粒体特异性标记探针Mitotracker Red结果显示老化卵母细胞内活性线粒体丰度明显减少;类似地,通过对线粒体基因组(mitochondrial DNA,mtDNA)拷贝数的绝对定量检测,发现随着H_2O_2处理浓度的升高,卵母细胞mtDNA拷贝数呈现逐渐下降的趋势;此外,使用JC-1探针对线粒体膜电位(mitochondrial membrane potential,MMP)检测结果显示,10~100μM浓度下表现出MMP升高的趋势,即超极化现象,这种现象是凋亡早期特征性事件,意味着老化卵母细胞已经启动了凋亡的发生。老化卵母细胞中氧化应激对IVF胚胎的持续影响,提示这一过程是可恢复的。因此,我们尝试在体外培养阶段(in vitro culture,IVC)添加褪黑素(melatonion,MT),通过清除附植前胚胎中的活性氧来缓解老化卵母细胞IVF后的发育损伤。选取了较为温和的100μM H_2O_2浓度诱导卵母细胞老化,分别在培养液中添加10-5 M、10-7 M、10-9 M三个浓度的褪黑素,评价老化卵母细胞IVF后发育效率和发育质量。结果显示,与老化组(20.87±4.11%)相比,培养液中添加10-9 M褪黑素可以显著恢复囊胚率(29.42±2.39%),达到与青年卵相当的发育水平(38.76±9.43%)。而10-5 M和10-7 M褪黑素添加则未能改善囊胚率(19.87±2.88%、23.35±6.03%)。这些结果表明IVF后培养阶段添加低浓度的褪黑素可以有效恢复和改善老化卵母细胞IVF后的发育能力。接下来,我们发现10-5 M、10-7M、10-9 M褪黑素添加后,囊胚细胞数分别为28.30±10.42%、31.54±9.97%、39.36±9.78%,均与H_2O_2处理老化组(37.91±4.25%)差异不显著。进一步,囊胚凋亡检测结果提示,10-9 M褪黑素添加有降低老化卵IVF囊胚细胞凋亡率的趋势,但差异不显著(2.57%vs.3.18%)。我们意外地发现,10-5 M褪黑素会显著增加囊胚细胞凋亡率(6.97%vs.3.18%,P0.05),提示高浓度的褪黑素添加会有细胞毒害作用。mtDNA定量结果显示,10-9 M褪黑素添加组与H_2O_2处理老化组相比,能够显著恢复8细胞阶段mtDNA拷贝数。结论:1)100μM H_2O_2诱导卵母细胞老化,卵母细胞内线粒体相关特征受损明显,其中线粒体中ROS水平显著升高,活性线粒体丰度和mtDNA拷贝数降低,膜电位超极化现象明显。2)在IVF后的IVC阶段添加10-9M褪黑素可以恢复H_2O_2引起的线粒体拷贝数降低,提高囊胚率及囊胚质量,从而缓解由H_2O_2诱导的老化卵母细胞体外受精后发育受阻。
[Abstract]:Elderly women undergoing in vitro fertilization (in vitro, fertilization, IVF) after the treatment success rate was significantly lower. The oocyte aging caused by the embryonic development ability is the result of elderly women after in vitro fertilization and low pregnancy rate, abortion rate an important factor. Therefore, looking for is of great significance to alleviate aging of oocytes after IVF cells blocked development. In this experiment, hydrogen peroxide (H_2O_2) treatment of oxidative stress in mouse oocytes established induced aging model as the research object, the relevant characteristics of the oxidative stress induced oocyte aging after mitochondrial impairment and decline rate and quality of embryos aged oocytes after IVF. Then, with aging oocytes to the blastocyst rate and blastocyst quality as the main index, and assisted in the mitochondria function, effects of melatonin to improve oocyte aging IVF embryo rate and quality The amount of the feasibility, and provide experimental basis for improving the clinical success rate of IVF in elderly women. In this study, using 10 M, 50 M, 100 M and 150 M were treated with different concentrations of H_2O_2 MII oocytes 3 h aging model on oxidative stress of oocytes, without H_2O_2 treated oocytes as controls. Each group of oocytes are standard IVF, observe the development efficiency. The results show that with the increase of H_2O_2 concentration, degree of oxidative stress in oocytes was significantly increased. The oocytes IVF 150 M after H_2O_2 treatment, the fragmentation rate compared to the control group increased significantly (7.77 + 4.78%vs.39.32 + 9.14%, P0.05); 100 M after treatment with H_2O_2 2 cell rate compared to the control group decreased significantly (45.63 + 2.6%vs.54.67 + 8.27%, P0.05); when IVF embryo development to the blastocyst stage, 50 M, 100 M treatment group blastocyst rate were 26.27 + 0.06%, 28.46 + 3.45%, significantly The lower than that of the control group (34.9 + 1.77%, P0.05). This shows that the aging of oxidative stress in oocytes, may IVF embryos have a sustained impact, resulting in growth damage. In many cell types, aging is often accompanied by the damage of mitochondrial function obviously, mitochondrial function so we H_2O_2 induced oocyte aging makes a special detection. The activity of mitochondria specific labeled probe Mitotracker Red results showed that the aging of oocytes activated mitochondrial abundance decreased significantly; similarly, the mitochondrial genome (mitochondrial DNA, mtDNA) for absolute quantification of copy number detection, found that with the increase of the concentration of H_2O_2, the copy number of mtDNA oocytes presented the trend of gradual decline; in addition, the use of JC-1 on mitochondrial membrane potential probe (mitochondrial membrane potential, MMP) test results showed that the performance of 10~ 100 M concentration of MM The increasing of P, namely the hyperpolarization phenomenon, this phenomenon is characteristic of early apoptotic events, mean oocyte aging has initiated apoptosis. The aging effects of oxidative stress in oocytes of IVF embryos, suggesting that this process is reversible. Therefore, we try to develop stage (in vitro in vitro culture, IVC) (Melatonion, MT) added melatonin, by scavenging reactive oxygen in the embryo before implantation to alleviate the injury of aging development oocytes after IVF. Select a more modest 100 M concentration of H_2O_2 induced oocyte aging, respectively in the medium supplemented with 10-5 M, 10-7 M. Melatonin 10-9 M three concentration, the evaluation of aging oocytes after IVF development efficiency and quality of development. The results show that with the aging group (20.87 + 4.11%) compared to the medium supplemented with 10-9 M melatonin can significantly restore the blastocyst rate (29.42 + 2.39%), and reached the green The eggs considerable development level (38.76 + 9.43%). And the addition of 10-5 M and 10-7 M melatonin is unable to improve the blastocyst rate (19.87 + 2.88%, 23.35 + 6.03%). These results show that IVF can effectively add after training stage recovery and improvement of aging oocytes after IVF development of melatonin low concentration. Then, we found that 10-5 M, 10-7M, adding 10-9 M melatonin, cell number of blastocyst was 28.30 + 10.42%, 31.54 + 9.97%, 39.36 + 9.78%, were treated with H_2O_2 (37.91 + 4.25%) aging group, the difference was not significant. Further, the blastocyst apoptosis detection results showed that 10-9 M melatonin decreased the apoptosis of oocyte aging add IVF blastocysts the cell rate trend, but the difference was not significant (2.57%vs.3.18%). We were surprised to find that the 10-5 M melatonin significantly increased the apoptosis rate of blastocysts (6.97%vs.3.18%, P0.05), suggesting that high concentrations of melatonin may be added with cell toxicity of.MtDNA set The amount of results showed that 10-9 M melatonin group and H_2O_2 treatment group added aging compared to the 8 cell stage can significantly restore the mtDNA copy number. Conclusion: 1) 100 M H_2O_2 induced oocyte aging, mitochondria related characteristics of oocytes was damaged, the level of ROS in mitochondria significantly increased, mitochondrial activity and mtDNA abundance the copy number decreased, the membrane potential hyperpolarization phenomenon obviously.2) added melatonin 10-9M in IVC phase after IVF can restore H_2O_2 induced mitochondrial copy number decreased, improve the blastocyst rate and blastocyst quality, so as to alleviate the aging of oocytes in vitro fertilization induced by H_2O_2 after the development was blocked.

【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R714.8

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