牛卵母細(xì)胞中的DNA雙鏈損傷和P21基因的相關(guān)性研究
發(fā)布時(shí)間：2018-09-04 17:36
【摘要】：P21參與哺乳動(dòng)物的減數(shù)分裂調(diào)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,從而抑制MPF的活性。P21與卵母細(xì)胞上的DNA雙鏈損傷之間有沒有關(guān)系至今未見報(bào)道。近期有研究報(bào)道體細(xì)胞上的P21能夠促進(jìn)DNA雙鏈損傷的修復(fù)。在卵母細(xì)胞上P21是否也有這樣的作用是本研究關(guān)注的核心問題。本研究以牛的卵母細(xì)胞為試驗(yàn)材料,利用本實(shí)驗(yàn)室儲存的pVenus-P21的真核表達(dá)載體,首先重復(fù)了以前的超表達(dá)和干擾實(shí)驗(yàn),確定了P21在牛卵母細(xì)胞成熟上發(fā)揮的重要作用。然后利用DNA雙鏈損傷制造試劑Zeocin,先在Hela細(xì)胞上做了個(gè)梯度試驗(yàn),看看對Hela細(xì)胞增殖率的影響,目的是為卵母細(xì)胞上Zeocin的使用濃度提供參考范圍。然后在牛卵母細(xì)胞上利用Zeocin制造DNA的雙鏈損傷,并用雙鏈損傷標(biāo)記γH2AX免疫熒光鑒定了損傷的成功形成。統(tǒng)計(jì)DNA雙鏈損傷對卵母細(xì)胞成熟率的影響,利用Q-PCR對DNA雙鏈損傷后同源重組、非同源重組、P21、P53等基因的表達(dá)變化情況做了統(tǒng)計(jì)。同時(shí),對裸卵和卵丘卵母細(xì)胞復(fù)合體對DNA雙鏈損傷試劑的敏感性做了研究。后面,我們運(yùn)用ATM的特異性抑制劑ku55933處理卵母細(xì)胞,觀察ATM和卵母細(xì)胞成熟的相關(guān)性,同時(shí)在ATM被抑制的情況下,檢測P21是否和卵母細(xì)胞質(zhì)量評價(jià)基因的表達(dá)有相關(guān)性,目的是確定P21表達(dá)量能否作為卵母細(xì)胞質(zhì)量評價(jià)的一個(gè)參考。研究取得以下結(jié)果:1.鑒定了本實(shí)驗(yàn)室保留的pVenus-P21的真核表達(dá)載體的正確性,并能夠在Hela細(xì)胞和牛卵母細(xì)胞上表達(dá)。通過在牛卵母細(xì)胞上超表達(dá)和干擾P21驗(yàn)證了,超表達(dá)P21,牛卵母細(xì)胞的成熟率下降,干擾P21牛卵母細(xì)胞的成熟率上升的實(shí)驗(yàn)現(xiàn)象,確定了P21在牛卵母細(xì)胞成熟上的重要作用。2利用DNA雙鏈損傷試劑Zeocin對Hela細(xì)胞增殖率的影響,大體確定了在卵母細(xì)胞上Zeocin的使用濃度范圍。利用雙鏈損傷的特異性標(biāo)記γH2AX的免疫熒光確定了雙鏈損傷的形成。發(fā)現(xiàn)牛的卵母細(xì)胞減數(shù)第一次分裂時(shí)期對低劑量DNA的雙鏈損傷有一定的耐受性,當(dāng)DNA的雙鏈損傷達(dá)到一定強(qiáng)度后,才能阻滯卵母細(xì)胞第一極體的排出。在DNA雙鏈損傷情況下不能成熟的卵母細(xì)胞中在遭遇DNA雙鏈損傷情況下,不能成熟的卵母細(xì)胞P21的表達(dá)量隨著損傷強(qiáng)度的增加,先是反應(yīng)不敏感,然后是表達(dá)量增加(與成熟率對應(yīng))。在遭遇DNA雙鏈損傷的情況下,ATM的相對表達(dá)量在未成熟卵母細(xì)胞中與P21趨勢相同(這時(shí)的ATM不是主導(dǎo)修復(fù)功能而是主導(dǎo)的P53-P21周期阻滯功能)在遭遇DNA雙鏈損傷的情況下,參與非同源重組的KU70在能成熟的卵母細(xì)胞中隨著強(qiáng)度的增加先升高后降低。不能成熟的卵母細(xì)胞中一直下降。在成熟的卵母細(xì)胞中P21一直維持較低水平;在未成熟的卵母細(xì)胞中P21的變化趨勢和ATM,P53相吻合。3.卵丘卵母細(xì)胞復(fù)合體對Zeocin損傷敏感度低(卵丘細(xì)胞對卵母細(xì)胞的保護(hù),使卵母細(xì)胞不應(yīng)答;卵丘細(xì)胞命運(yùn)將走向凋亡,自身不應(yīng)答)4.ATM功能和減數(shù)第一次分裂的完成有十分密切的關(guān)系,當(dāng)ATM受到抑制時(shí),卵母細(xì)胞成熟率下降;在ku55933(ATM)的特異性抑制劑的作用下未成熟的卵母細(xì)胞中GDF-9,BMP-15(卵母細(xì)胞評價(jià)因子)有下調(diào)趨勢,H1foo與它們的趨勢相反;在成熟的卵母細(xì)胞中FSHR上調(diào)出現(xiàn)顯著性差異。在ku55933作用下,未成熟的卵母細(xì)胞中的P21有下調(diào)的趨勢。本研究得到如下結(jié)論:牛卵母細(xì)胞的減數(shù)第一次分裂進(jìn)程對DNA雙鏈損傷有一定的耐受性,當(dāng)損傷超過一定范圍時(shí)成熟率下降,下降的原因推測是ATM作用于P53-P21影響減數(shù)分裂的完成減數(shù)第一次分裂過程中遭遇DNA雙鏈損傷是非同源重組的KU70表達(dá)明顯提高,推測非同源重組在減數(shù)第一次分裂應(yīng)對DSBs中發(fā)揮著作用。卵丘卵母細(xì)胞復(fù)合體相對卵母細(xì)胞更耐受DNA雙鏈損傷試劑的作用,卵丘顆粒細(xì)胞在減數(shù)第一次分裂前期發(fā)揮著重要的作用。ATM活性對卵母細(xì)胞減數(shù)第一次分裂的完成具有重要意義,當(dāng)ATM被抑制后成熟率下降。在ATM被抑制的情況下,未成熟的卵母細(xì)胞中H1ffo的趨勢和GDF-9,BMP-1相反。ATM抑制劑的作用下,成熟的卵母細(xì)胞FSHR上調(diào)明顯。
[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.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S814.1
本文編號：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.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S814.1
【相似文獻(xiàn)】
相關(guān)碩士學(xué)位論文 前1條
1 許曉磊;牛卵母細(xì)胞中的DNA雙鏈損傷和P21基因的相關(guān)性研究[D];西北農(nóng)林科技大學(xué);2015年
，本文編號：2222871
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