本文選題：卵母細(xì)胞老化　切入點(diǎn)：H_2O_2　出處：《中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：高齡婦女接受體外受精(in vitro fertilization,IVF)治療后的成功率明顯偏低。其中,卵母細(xì)胞老化造成的胚胎發(fā)育能力下降,是導(dǎo)致高齡婦女體外受精后妊娠率低、流產(chǎn)率高的重要原因。因此,尋找能夠緩解老化卵母細(xì)胞IVF后發(fā)育受阻的方法具有重要的意義。本實(shí)驗(yàn)以過(guò)氧化氫(H_2O_2)處理小鼠卵母細(xì)胞建立的氧化應(yīng)激誘導(dǎo)老化模型為研究對(duì)象,研究氧化應(yīng)激誘導(dǎo)卵母細(xì)胞老化后線粒體的相關(guān)特征受損與老化卵母細(xì)胞IVF后胚胎發(fā)育率及質(zhì)量下降的關(guān)系。繼而,以老化卵母細(xì)胞囊胚發(fā)育率和囊胚質(zhì)量為主要評(píng)價(jià)指標(biāo),并輔于線粒體功能狀態(tài),考察褪黑素改善老化卵母細(xì)胞IVF胚胎發(fā)育率及質(zhì)量的可行性,為提高臨床高齡女性IVF成功率提供實(shí)驗(yàn)依據(jù)。本研究分別利用10μM、50μM、100μM和150μM不同濃度的H_2O_2處理MII期卵母細(xì)胞3 h,建立以氧化應(yīng)激為特征的卵母細(xì)胞老化模型,以未經(jīng)H_2O_2處理的卵母細(xì)胞為對(duì)照。各組卵母細(xì)胞進(jìn)行標(biāo)準(zhǔn)的IVF后,觀察發(fā)育效率。結(jié)果顯示,隨H_2O_2濃度的增加,卵母細(xì)胞中的氧化應(yīng)激程度是明顯加劇的。經(jīng)150μM H_2O_2處理的卵母細(xì)胞IVF后,碎裂率相比對(duì)照組顯著性升高(7.77±4.78%vs.39.32±9.14%,P0.05);100μM H_2O_2處理后2細(xì)胞率相比對(duì)照組顯著降低(45.63±2.6%vs.54.67±8.27%,P0.05);當(dāng)IVF胚胎發(fā)育至囊胚階段時(shí),50μM、100μM處理組囊胚率分別為26.27±0.06%、28.46±3.45%,均顯著低于對(duì)照組(34.9±1.77%,P0.05)。這說(shuō)明老化卵母細(xì)胞中的氧化應(yīng)激,可能會(huì)對(duì)IVF后胚胎產(chǎn)生持續(xù)的影響,造成發(fā)育損傷。在許多細(xì)胞類型中,老化往往伴隨著明顯的線粒體功能損傷,因此我們對(duì)H_2O_2誘導(dǎo)卵母細(xì)胞老化的線粒體功能進(jìn)行了專門的檢測(cè)。活性線粒體特異性標(biāo)記探針Mitotracker Red結(jié)果顯示老化卵母細(xì)胞內(nèi)活性線粒體豐度明顯減少;類似地,通過(guò)對(duì)線粒體基因組(mitochondrial DNA,mtDNA)拷貝數(shù)的絕對(duì)定量檢測(cè),發(fā)現(xiàn)隨著H_2O_2處理濃度的升高,卵母細(xì)胞mtDNA拷貝數(shù)呈現(xiàn)逐漸下降的趨勢(shì);此外,使用JC-1探針對(duì)線粒體膜電位(mitochondrial membrane potential,MMP)檢測(cè)結(jié)果顯示,10~100μM濃度下表現(xiàn)出MMP升高的趨勢(shì),即超極化現(xiàn)象,這種現(xiàn)象是凋亡早期特征性事件,意味著老化卵母細(xì)胞已經(jīng)啟動(dòng)了凋亡的發(fā)生。老化卵母細(xì)胞中氧化應(yīng)激對(duì)IVF胚胎的持續(xù)影響,提示這一過(guò)程是可恢復(fù)的。因此,我們嘗試在體外培養(yǎng)階段(in vitro culture,IVC)添加褪黑素(melatonion,MT),通過(guò)清除附植前胚胎中的活性氧來(lái)緩解老化卵母細(xì)胞IVF后的發(fā)育損傷。選取了較為溫和的100μM H_2O_2濃度誘導(dǎo)卵母細(xì)胞老化,分別在培養(yǎng)液中添加10-5 M、10-7 M、10-9 M三個(gè)濃度的褪黑素,評(píng)價(jià)老化卵母細(xì)胞IVF后發(fā)育效率和發(fā)育質(zhì)量。結(jié)果顯示,與老化組(20.87±4.11%)相比,培養(yǎng)液中添加10-9 M褪黑素可以顯著恢復(fù)囊胚率(29.42±2.39%),達(dá)到與青年卵相當(dāng)?shù)陌l(fā)育水平(38.76±9.43%)。而10-5 M和10-7 M褪黑素添加則未能改善囊胚率(19.87±2.88%、23.35±6.03%)。這些結(jié)果表明IVF后培養(yǎng)階段添加低濃度的褪黑素可以有效恢復(fù)和改善老化卵母細(xì)胞IVF后的發(fā)育能力。接下來(lái),我們發(fā)現(xiàn)10-5 M、10-7M、10-9 M褪黑素添加后,囊胚細(xì)胞數(shù)分別為28.30±10.42%、31.54±9.97%、39.36±9.78%,均與H_2O_2處理老化組(37.91±4.25%)差異不顯著。進(jìn)一步,囊胚凋亡檢測(cè)結(jié)果提示,10-9 M褪黑素添加有降低老化卵IVF囊胚細(xì)胞凋亡率的趨勢(shì),但差異不顯著(2.57%vs.3.18%)。我們意外地發(fā)現(xiàn),10-5 M褪黑素會(huì)顯著增加囊胚細(xì)胞凋亡率(6.97%vs.3.18%,P0.05),提示高濃度的褪黑素添加會(huì)有細(xì)胞毒害作用。mtDNA定量結(jié)果顯示,10-9 M褪黑素添加組與H_2O_2處理老化組相比,能夠顯著恢復(fù)8細(xì)胞階段mtDNA拷貝數(shù)。結(jié)論:1)100μM H_2O_2誘導(dǎo)卵母細(xì)胞老化,卵母細(xì)胞內(nèi)線粒體相關(guān)特征受損明顯,其中線粒體中ROS水平顯著升高,活性線粒體豐度和mtDNA拷貝數(shù)降低,膜電位超極化現(xiàn)象明顯。2)在IVF后的IVC階段添加10-9M褪黑素可以恢復(fù)H_2O_2引起的線粒體拷貝數(shù)降低,提高囊胚率及囊胚質(zhì)量,從而緩解由H_2O_2誘導(dǎo)的老化卵母細(xì)胞體外受精后發(fā)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.

【學(xué)位授予單位】：中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R714.8

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本文編號(hào)：1631998