【作者】 陈曼；

【导师】 李志凌；

【作者基本信息】 汕头大学 ， 临床医学， 2011， 硕士

【摘要】 前言DNA双链断裂（DNA double-stranded breaks ,DSBs）发生后,哺乳动物细胞将启动一个复杂的蛋白网络对其进行监控及修复:包括细胞周期检验点、DNA损伤修复及凋亡。DNA损伤反应是高度保守的信号感应过程,大致可分为损伤感应、信号传递及信号效应3个部分。损伤感应分子3-磷脂酰肌醇激酶（Phosphoinositide 3-kinaserelated protein kinase,PI-3K）家族成员针对不同的损伤来源被激活后,迅速聚集到损伤位点,快速磷酸化下游的效应激酶,通过协同作用实现对DNA损伤的高度协调反应。H2AX作为PI-3K家族成员的下游分子,其第139位丝氨酸残基被快速磷酸化后形成γH2AX。后者被认为是DNA损伤修复标志物,而ATM所具有的可被DNA损伤相关的染色质修饰所激活的能力,使其成为PI-3K家族中促使H2AX磷酸化最匹配的激酶。同时,ATM也参与了细胞周期检验点的调控:激活的ATM磷酸化细胞周期检验点激酶Chk1或Chk2,激活的Chk1/Chk2进而顺序磷酸化不同的效应因子,并根据DNA的损伤类型,形成不同的信号通路激活细胞周期检验点（G1/S、S、G2/M）,使细胞周期停滞,为DNA损伤修复提供时间。精液冷冻保存是辅助生殖技术（Assisted reproductive technology,ART）领域中的重要技术,其冻融过程产生过量的活性氧类物质（Reactive oxygen species,ROS）可致精子氧化应激性DNA损伤。ART应用又使DNA损伤精子受精发育成胚胎的机会增加,尤其是卵细胞浆内单精子注射技术（Intra-cytoplasmic sperm injection,ICSI）,由于避开了自然受精过程中多种天然屏障对精子的筛选,增加了DNA损伤精子直接进入卵子的风险。DNA损伤精子仍可受精,但成熟精子丧失自我修复DNA损伤的能力,需依赖受精后受精卵内的修复。而目前关于氧化应激性DNA损伤精子受精后受精卵修复的研究十分有限,进行该方向的研究为完善DNA损伤精子受精后受精卵修复理论无疑具有重大意义。正是由于精子氧化应激性DNA损伤与ROS密切相关,在ART体系中添加抗氧化保护剂以降低ROS引发的氧化应激性损伤可能是提高ART治疗结局的有效途径之一。没食子儿茶素没食子酸酯（Epigallocatechin-3-gallate ,EGCG）因具有非常强的抗氧化活性,能够保护细胞和DNA免受损害而作为目前众多抗氧化保护剂中最理想的一种,被尝试添加到体外受精培养体系中,表现出促进胚胎发育的作用,但是其对冻融精子受精卵的保护机制尚不清楚。阐明EGCG对氧化应激性DNA损伤精子受精后受精卵的保护机制,不仅为研究冷冻精子氧化应激性DNA损伤防治措施提供理论依据,也为寻找降低DNA损伤精子遗传风险的策略方法提供了新思路。目的1.初步探讨氧化应激性DNA损伤精子受精后受精卵DNA损伤修复机制。2.初步探讨EGCG对氧化应激性DNA损伤精子受精后受精卵的保护机制。方法1.分别将昆明（KunMing,KM）小鼠精子放入新鲜精子获能液、加1mMH₂O₂的精子获能液中,5%CO₂、37℃培养箱孵育1.5小时左右,得到新鲜精子和氧化应激性DNA损伤精子,调整精子浓度为1-2.5×10⁶个/ml,分别进行体外受精获得正常对照组及氧化应激性DNA损伤组受精卵。采用免疫荧光法检测正常对照组及氧化应激性DNA损伤组原核期胚胎中pSer1981-ATM、pSer345-Chk1及pThr68-Chk2焦点的形成并进行比较。2.依据受精液和胚胎培养液中是否添加EGCG,将氧化应激性DNA损伤组受精卵分两组:未添加组受精液和胚胎培养液中未添加EGCG;添加组受精液和胚胎培养液中均添加了17.5μg/ml EGCG。从受精后16.5小时至23.5小时,分别对未添加组及添加组的受精率、二细胞形成情况及四细胞形成情况进行观察,每小时观察1次,计算受精率、一细胞卵裂率及二细胞卵裂率并进行比较;选择未添加组及添加组上述各观察时间点的受精卵,免疫荧光法检测pSer1981-ATM焦点的形成并进行比较。结果1.免疫荧光法检测各组受精卵中pSer1981-ATM信号,发现正常对照组受精卵中pSer1981-ATM、pSer345-Chk1及pThr68-Chk2均无信号;氧化应激性DNA损伤组中可见较强的pSer1981-ATM荧光信号及pSer345-Chk1荧光信号,pThr68-Chk2则未见明显信号。2.未添加组的受精率、一细胞卵裂率及二细胞卵裂率分别为45.2%、100%及61.5%,添加组的受精率、一细胞卵裂率及二细胞卵裂率分别为44.5%、97.8%及71.3%,添加组与未添加组相比,受精率、一细胞卵裂率及二细胞卵裂率差别均无统计学意义（P>0.05）。从受精后16.5小时至受精后23.5小时,未添加组的一细胞卵裂率依次是21%、34%、68%、86%、89%、91%、96%、98%;添加组的一细胞卵裂率依次是32%、70%、83%、87%、89%、90%、95%、96%;对各个时间点添加组及未添加组的一细胞卵裂率进行比较发现,受精后17.5小时和受精后18.5小时（相当于第一次有丝分裂细胞周期的晚G2期）的一细胞卵裂率差别有统计学意义（P<0.05）,其它时间点的一细胞卵裂率差别无统计学意义（P>0.05）。以受精后时间为自变量,一细胞卵裂率为因变量建立函数,以一细胞卵裂率为50%的时间点作为观测点,当一细胞胚胎卵裂率为50%时,未添加组为受精后18.28598小时,添加组为受精后17.10280小时,添加组比未添加组提前约1小时进入分裂。未添加组和添加组pSer1981-ATM荧光信号强度分别为0.008327±0.006603和0.0196±0.010347,差别有统计学意义（P<0.05）。结论1.氧化应激性DNA损伤精子受精后DNA损伤的修复依赖于卵母细胞中储存的mRNA和蛋白质,可能通过ATM启动了受精卵DNA损伤修复机制的同时,ATM还可能激活Chk1启动细胞周期检验点,使细胞分裂发生延迟,从而为修复赢得时间。2. EGCG主要作用于受精后第一个细胞周期,加快受精卵进入卵裂的速度、延长第一次卵裂的持续时间;EGCG可能促进了氧化应激性DNA损伤精子受精后受精卵内ATM介导的损伤修复。更多还原

【Abstract】 ForewordMammalian cell will initiate a complex protein’network in response to DNA double-stranded breaks （DSBs）, which includes cell cycle checkpoint, DNA damage repair and apoptosis. DNA damage signal transduction pathway is a highly conserved one, which could be devided into three parts: sensors, transducers and effectors. Phosphoinositide 3-kinaserelated protein kinases （PI-3K） family are activated according to different DNA damage sources and quickly recruited to the damage site.It phosphorylates downstream kinases, which work cooperatively in order to carry out the concordant cellular responses to DNA damage, including H2AX, which is rapidly phosphorylated at Ser139 to formγH2AX once DSBs occurs and is characterized as the marker of DNA damage. Ataxia telangiectasia mutated （ATM） can be activated by the chromatin modification associated with DNA damage, making it the most match kinase for H2AX phosphorylation of PI-3K family. ATM also regulates cell cycle checkpoint and apoptosis: Activated ATM phosphorylates and activates cell cycle checkpoint kinases Chk1 and Chk2, mediates cascade reaction and amplifys damage signal.Chk1/Chk2 phosphorylates its effectors to activate cell cycle checkpoints （G1/S, S, and G2/M） by different signaling pathways according to different DNA sources, so as to stop the cell cycle and provide time for DNA repair.Semen cryopreservation is an very important technology in assisted reproductive technology （ART） field. But it could produces excessive reactive oxygen species （ROS） during frozen-thawed process and induces oxidative damge on sperm.With the application of ART, especially intra-cytoplasmic sperm injection（ICSI）,which avoids multiple natural barriers for sperm screening, the opportunity raises for DNA-damge sperm to fertilize and develop into embryo. DNA-damage sperm still can fertilize, while it is unable to repair damaged DNA for mature sperm, and depends on the repair of zygotes after fertilization. However, the research on repair in zygote fertilized with oxidative stress-induced DNA-damage sperm is limited.Because of the association between oxidative stress-induced DNA-damage and ROS, it may be beneficial for ART outcomes by adding antioxidant into the ART system to reduce the DNA damage caused by ROS. Epigallocatechin-3-gallate （EGCG） is the best candidate due to its strongly anti-oxidation activity and protective effect on cell and DNA. It was added to the ART system and found it could promote embryo development, but the mechanism remains unknown.Objective1. To explore repair mechanism in zygote fertilized with oxidative stress-induced DNA-damage sperm.2. To explore protective mechanism of EGCG on zygote fertilized with oxidative stress-induced DNA-damage sperm.Methods1. KM mouse sperm was added to capacitation agent of fresh or 1mM H₂O₂ for capacity in 5%CO₂,37℃incubator for 1.5 hours,so as to get fresh and oxidative DNA-damage sperm for IVF, adjusting the concentration of sperm to 1-2.5×106/ml. It was devided into two groups: the control group without H₂O₂ in capacitation agent and the oxidative DNA -damage group with 1mM H₂O₂ in capacitation agent, pSer1981-ATM ,pSer345-Chk1 and pThr68-Chk2 signaling in zygotes of pronucleus stage was detected by immunofluorescence and analyzed.2. The zygotes fertilized with oxidative DNA-damage sperm were got from the first part and divided into two groups: the blank group without EGCG neither in insemination agent nor development agent; the experimental group with 17.5μg/ml EGCG both in insemination agent and development agent. Zogotes of above two groups were observed every one hour from 16.5 hours post-insemination（hpi） to 23.5 hpi and analyzed their fertilization rate,one- and two-cell cleavage rate. PSer1981-ATM signaling in zygotes of above the observation time between the blank group and the experimental group were detected by immunofluorescence and analyzed. Results1. PSer1981-ATM and pSer345-Chk1 signaling were found in zygote with treated group while no positive result was found in the control group. No pThr68-Chk2 signaling was found in neither the control group nor the oxidative DNA-damage group.2. The fertilization rate, the cleavage rate of one- and two-cell embryos was 44.5%、97.8% and 71.3% respectively of the experimental group, 45.2%、100% and 61.5% respectively of the blank group,there was no statistical significance for fertilization rate, one- and two-cell cleavage rate between them（P>0.05）.The cleavage rate of one-cell embryos of the experimental group was 32%、70%、83%、87%、89%、90%、95%、96% respectively from 16.5 hpi to 23.5 hpi,while the cleavage rate of one-cell embryos of the blank group was 21%、34%、68%、86%、89%、91%、96%、98% respectively. The cleavage rate of one-cell embryos at the same observation time between the blank group and the experimental group was analyzed and found that there was statistical significance for them when at 17.5hpi and 18.5hpi（P<0.05）,while there was no statistical significance at the other observation time （P>0.05）.The zygotes in the experimental group entered M phase about one hour earlier when determined as the time at which 50% of the embyros had cleaved. PSer1981-ATM signaling was found in the experimental group and it was stronger than that in the blank group, fluorescence analysis showed the intensity was 0.0196±0.010347 and 0.008327±0.006603 for the experimental group and the blank group respectively,there was statistical significance（P<0.05）.Conclusions1. It relied on the mRNA and proteins stored in oocytes for the repair of oxidative DNA-damage sperm post fertilization. It might be ATM that initiated the DNA damage repair mechanism in zygotes; meanwhile, ATM might activate Chk1, initiate the cell cycle checkpoint and delay cell division, so as to gain time for repair.2. It was possible that EGCG mediated the DNA damage repair mechanism by accelerating the speed for entering M phase and prolonging the duration of M phase; EGCG might promote the repair of zygote fertilized with oxidative DNA-damage sperm which mediated by ATM更多还原