牛卵母细胞中的DNA双链损伤和P21基因的相关性研究
发布时间：2018-09-04 17:36
【摘要】：P21参与哺乳动物的减数分裂调控的研究,以前主要集中在P21可以直接抑制CDK1激酶的磷酸化,从而抑制MPF的活性。P21与卵母细胞上的DNA双链损伤之间有没有关系至今未见报道。近期有研究报道体细胞上的P21能够促进DNA双链损伤的修复。在卵母细胞上P21是否也有这样的作用是本研究关注的核心问题。本研究以牛的卵母细胞为试验材料,利用本实验室储存的pVenus-P21的真核表达载体,首先重复了以前的超表达和干扰实验,确定了P21在牛卵母细胞成熟上发挥的重要作用。然后利用DNA双链损伤制造试剂Zeocin,先在Hela细胞上做了个梯度试验,看看对Hela细胞增殖率的影响,目的是为卵母细胞上Zeocin的使用浓度提供参考范围。然后在牛卵母细胞上利用Zeocin制造DNA的双链损伤,并用双链损伤标记γH2AX免疫荧光鉴定了损伤的成功形成。统计DNA双链损伤对卵母细胞成熟率的影响,利用Q-PCR对DNA双链损伤后同源重组、非同源重组、P21、P53等基因的表达变化情况做了统计。同时,对裸卵和卵丘卵母细胞复合体对DNA双链损伤试剂的敏感性做了研究。后面,我们运用ATM的特异性抑制剂ku55933处理卵母细胞,观察ATM和卵母细胞成熟的相关性,同时在ATM被抑制的情况下,检测P21是否和卵母细胞质量评价基因的表达有相关性,目的是确定P21表达量能否作为卵母细胞质量评价的一个参考。研究取得以下结果:1.鉴定了本实验室保留的pVenus-P21的真核表达载体的正确性,并能够在Hela细胞和牛卵母细胞上表达。通过在牛卵母细胞上超表达和干扰P21验证了,超表达P21,牛卵母细胞的成熟率下降,干扰P21牛卵母细胞的成熟率上升的实验现象,确定了P21在牛卵母细胞成熟上的重要作用。2利用DNA双链损伤试剂Zeocin对Hela细胞增殖率的影响,大体确定了在卵母细胞上Zeocin的使用浓度范围。利用双链损伤的特异性标记γH2AX的免疫荧光确定了双链损伤的形成。发现牛的卵母细胞减数第一次分裂时期对低剂量DNA的双链损伤有一定的耐受性,当DNA的双链损伤达到一定强度后,才能阻滞卵母细胞第一极体的排出。在DNA双链损伤情况下不能成熟的卵母细胞中在遭遇DNA双链损伤情况下,不能成熟的卵母细胞P21的表达量随着损伤强度的增加,先是反应不敏感,然后是表达量增加(与成熟率对应)。在遭遇DNA双链损伤的情况下,ATM的相对表达量在未成熟卵母细胞中与P21趋势相同(这时的ATM不是主导修复功能而是主导的P53-P21周期阻滞功能)在遭遇DNA双链损伤的情况下,参与非同源重组的KU70在能成熟的卵母细胞中随着强度的增加先升高后降低。不能成熟的卵母细胞中一直下降。在成熟的卵母细胞中P21一直维持较低水平;在未成熟的卵母细胞中P21的变化趋势和ATM,P53相吻合。3.卵丘卵母细胞复合体对Zeocin损伤敏感度低(卵丘细胞对卵母细胞的保护,使卵母细胞不应答;卵丘细胞命运将走向凋亡,自身不应答)4.ATM功能和减数第一次分裂的完成有十分密切的关系,当ATM受到抑制时,卵母细胞成熟率下降;在ku55933(ATM)的特异性抑制剂的作用下未成熟的卵母细胞中GDF-9,BMP-15(卵母细胞评价因子)有下调趋势,H1foo与它们的趋势相反;在成熟的卵母细胞中FSHR上调出现显著性差异。在ku55933作用下,未成熟的卵母细胞中的P21有下调的趋势。本研究得到如下结论:牛卵母细胞的减数第一次分裂进程对DNA双链损伤有一定的耐受性,当损伤超过一定范围时成熟率下降,下降的原因推测是ATM作用于P53-P21影响减数分裂的完成减数第一次分裂过程中遭遇DNA双链损伤是非同源重组的KU70表达明显提高,推测非同源重组在减数第一次分裂应对DSBs中发挥着作用。卵丘卵母细胞复合体相对卵母细胞更耐受DNA双链损伤试剂的作用,卵丘颗粒细胞在减数第一次分裂前期发挥着重要的作用。ATM活性对卵母细胞减数第一次分裂的完成具有重要意义,当ATM被抑制后成熟率下降。在ATM被抑制的情况下,未成熟的卵母细胞中H1ffo的趋势和GDF-9,BMP-1相反。ATM抑制剂的作用下,成熟的卵母细胞FSHR上调明显。
[Abstract]:P21 is involved in the regulation of meiosis in mammals. Previous studies have focused on P21 directly inhibiting the phosphorylation of CDK1 kinase and thus inhibiting the activity of MPF. In this study, bovine oocytes were used as experimental materials, and the eukaryotic expression vectors of pVenus-P21 stored in our laboratory were used to repeat the previous overexpression and interference experiments to determine the important role of P21 in bovine oocyte maturation. Then Zeocin, a DNA double-strand damage manufacturing reagent, was used to do a gradient test on Hela cells to see the effect on the proliferation rate of Hela cells. The purpose was to provide a reference range for the concentration of Zeocin on oocytes. Then Zeocin was used to make DNA double-strand damage on bovine oocytes and double-strand damage was used to label gamma H2AX immunofluorescence. The effects of DNA double strand damage on the maturation rate of oocytes were analyzed. The expression of homologous recombination, non-homologous recombination, P21, P53 and other genes after DNA double strand damage were analyzed by Q-PCR. Meanwhile, the sensitivity of nude and cumulus oocyte complexes to DNA double strand damage reagents was studied. We used the specific inhibitor of ATM, ku55933, to observe the correlation between ATM and oocyte maturation. At the same time, we detected the correlation between P21 and the expression of oocyte quality assessment gene when ATM was inhibited. The following results were obtained: 1. The eukaryotic expression vector of pVenus-P21 retained in our laboratory was identified and expressed in Hela cells and bovine oocytes. Phenomenon confirmed the important role of P21 in bovine oocyte maturation. 2 The concentration range of Zeocin on oocytes was determined by the effect of DNA double strand damage reagent Zeocin on the proliferation rate of Hela cells. The formation of double strand damage was determined by the immunofluorescence of double strand damage specific marker gamma H2AX. During the first division of meiosis, there is a certain tolerance to the double strand damage of low dose DNA. When the double strand damage of DNA reaches a certain intensity, the first polar body of oocytes can be blocked. The relative expression of ATM in immature oocytes tends to be the same as that in P21 (ATM is not the dominant repair function but the dominant cycle blocking function of P53-P21) in the case of DNA double strand damage. In the case of strand damage, KU70 involved in non-homologous recombination increased first and then decreased with the increase of strength in mature oocytes. In immature oocytes, P21 remained at a lower level; in immature oocytes, P21 trend was consistent with ATM, P53. 3. cumulus oocytes Maternal cell complex has a low sensitivity to Zeocin damage (cumulus cells protect the oocyte from responding; cumulus cells fate towards apoptosis, not responding to itself). 4. ATM function is closely related to the completion of the first meiotic division. When ATM is inhibited, oocyte maturation rate decreases; in ku55933 (ATM) characteristics GDF-9 and BMP-15 (oocyte evaluation factor) in immature oocytes were down-regulated by heterosexual inhibitors, but H1foo was opposite to them. FSHR was up-regulated significantly in mature oocytes. P21 in immature oocytes was down-regulated by ku55933. The first meiotic division of bovine oocytes was tolerant to DNA double strand damage. The maturation rate decreased when the damage exceeded a certain range. The reason for the decrease was presumed to be that the expression of homologous recombinant KU70 increased significantly when ATM acted on P53-P21 during the first meiotic division. The cumulus oocyte complex is more tolerant to DNA double strand damage reagents than the oocyte. The cumulus granulosa cells play an important role in the prophase of the first meiotic division. ATM activity plays an important role in the completion of the first meiotic division of the oocyte. When ATM was inhibited, the maturation rate decreased. When ATM was inhibited, the tendency of H1ffo in immature oocytes was opposite to that of GDF-9 and BMP-1. Under the action of ATM inhibitors, FSHR in mature oocytes was up-regulated significantly.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S814.1
本文编号：2222871
[Abstract]:P21 is involved in the regulation of meiosis in mammals. Previous studies have focused on P21 directly inhibiting the phosphorylation of CDK1 kinase and thus inhibiting the activity of MPF. In this study, bovine oocytes were used as experimental materials, and the eukaryotic expression vectors of pVenus-P21 stored in our laboratory were used to repeat the previous overexpression and interference experiments to determine the important role of P21 in bovine oocyte maturation. Then Zeocin, a DNA double-strand damage manufacturing reagent, was used to do a gradient test on Hela cells to see the effect on the proliferation rate of Hela cells. The purpose was to provide a reference range for the concentration of Zeocin on oocytes. Then Zeocin was used to make DNA double-strand damage on bovine oocytes and double-strand damage was used to label gamma H2AX immunofluorescence. The effects of DNA double strand damage on the maturation rate of oocytes were analyzed. The expression of homologous recombination, non-homologous recombination, P21, P53 and other genes after DNA double strand damage were analyzed by Q-PCR. Meanwhile, the sensitivity of nude and cumulus oocyte complexes to DNA double strand damage reagents was studied. We used the specific inhibitor of ATM, ku55933, to observe the correlation between ATM and oocyte maturation. At the same time, we detected the correlation between P21 and the expression of oocyte quality assessment gene when ATM was inhibited. The following results were obtained: 1. The eukaryotic expression vector of pVenus-P21 retained in our laboratory was identified and expressed in Hela cells and bovine oocytes. Phenomenon confirmed the important role of P21 in bovine oocyte maturation. 2 The concentration range of Zeocin on oocytes was determined by the effect of DNA double strand damage reagent Zeocin on the proliferation rate of Hela cells. The formation of double strand damage was determined by the immunofluorescence of double strand damage specific marker gamma H2AX. During the first division of meiosis, there is a certain tolerance to the double strand damage of low dose DNA. When the double strand damage of DNA reaches a certain intensity, the first polar body of oocytes can be blocked. The relative expression of ATM in immature oocytes tends to be the same as that in P21 (ATM is not the dominant repair function but the dominant cycle blocking function of P53-P21) in the case of DNA double strand damage. In the case of strand damage, KU70 involved in non-homologous recombination increased first and then decreased with the increase of strength in mature oocytes. In immature oocytes, P21 remained at a lower level; in immature oocytes, P21 trend was consistent with ATM, P53. 3. cumulus oocytes Maternal cell complex has a low sensitivity to Zeocin damage (cumulus cells protect the oocyte from responding; cumulus cells fate towards apoptosis, not responding to itself). 4. ATM function is closely related to the completion of the first meiotic division. When ATM is inhibited, oocyte maturation rate decreases; in ku55933 (ATM) characteristics GDF-9 and BMP-15 (oocyte evaluation factor) in immature oocytes were down-regulated by heterosexual inhibitors, but H1foo was opposite to them. FSHR was up-regulated significantly in mature oocytes. P21 in immature oocytes was down-regulated by ku55933. The first meiotic division of bovine oocytes was tolerant to DNA double strand damage. The maturation rate decreased when the damage exceeded a certain range. The reason for the decrease was presumed to be that the expression of homologous recombinant KU70 increased significantly when ATM acted on P53-P21 during the first meiotic division. The cumulus oocyte complex is more tolerant to DNA double strand damage reagents than the oocyte. The cumulus granulosa cells play an important role in the prophase of the first meiotic division. ATM activity plays an important role in the completion of the first meiotic division of the oocyte. When ATM was inhibited, the maturation rate decreased. When ATM was inhibited, the tendency of H1ffo in immature oocytes was opposite to that of GDF-9 and BMP-1. Under the action of ATM inhibitors, FSHR in mature oocytes was up-regulated significantly.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S814.1
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1 许晓磊;牛卵母细胞中的DNA双链损伤和P21基因的相关性研究[D];西北农林科技大学;2015年
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