本文選題：褪黑激素 + 豬卵母細(xì)胞　； 參考：《西南大學(xué)》2015年碩士論文

【摘要】：褪黑激素(MT)是一種吲哚類激素,在人及哺乳動(dòng)物體內(nèi)主要是由松果腺分泌,其他器官和細(xì)胞也有少量分泌,分泌量受光照影響比較大,且呈現(xiàn)晝夜節(jié)律性,一般夜晚分泌多,白天分泌少。MT能夠透過(guò)細(xì)胞膜及各種膜結(jié)構(gòu)進(jìn)入細(xì)胞器來(lái)發(fā)揮抗氧化作用。目前MT的抗氧化、抗凋亡作用在小鼠、綿羊、雞等動(dòng)物卵母細(xì)胞體外成熟及早期胚胎中均有報(bào)道,但在豬卵母細(xì)胞體外成熟中報(bào)道較少。本研究以豬為試驗(yàn)對(duì)象,研究了褪黑激素對(duì)卵母細(xì)胞體外成熟及胚胎發(fā)育的影響。主要內(nèi)容如下：1褪黑激素對(duì)豬卵母細(xì)胞體外發(fā)育的影響(1)褪黑激素對(duì)豬卵母細(xì)胞體外成熟的影響挑選合格的卵母細(xì)胞,然后隨機(jī)分成6組,在添加褪黑激素(分別為0M、10-11M、10-9M、10-7M、10-5M、10-3M)的體外成熟培養(yǎng)液中培養(yǎng)46h,統(tǒng)計(jì)各組卵母細(xì)胞的極體排出率。結(jié)果發(fā)現(xiàn),10-9M組(77.54%)和10-7M組(69.2%)的極體率顯著高于對(duì)照組(67.81%)和其他試驗(yàn)組,該兩組之間差異不顯著。(2)褪黑激素對(duì)豬卵母細(xì)胞孤雌胚胎發(fā)育的影響挑選合格的卵母細(xì)胞置于未添加褪黑激素的體外成熟液中培養(yǎng)46h,接著進(jìn)行孤雌激活并隨機(jī)將卵母細(xì)胞放到6組添加褪黑激素(分別為0M、10-1M、10-9M、10-7M、10-5M、10-3M)的胚胎培養(yǎng)液中繼續(xù)培養(yǎng)。胚胎培養(yǎng)的第2d統(tǒng)計(jì)卵裂率,第7d統(tǒng)計(jì)囊胚率。結(jié)果發(fā)現(xiàn),褪黑激素未能提高卵母細(xì)胞孤雌發(fā)育的卵裂率,對(duì)照組和各試驗(yàn)組間差異不顯著。10-SM組(20.31%)的囊胚率顯著高于對(duì)照組(9.45%)和10-3M組(10.68%)。(3)褪黑激素對(duì)豬卵母細(xì)胞體外發(fā)育的影響成熟液和胚胎培養(yǎng)液分別添加10-9M、10-5M褪黑激素,卵母細(xì)胞體外培養(yǎng)46h后統(tǒng)計(jì)卵裂率。孤雌激活后繼續(xù)胚胎培養(yǎng),第2d統(tǒng)計(jì)卵裂率,第7d統(tǒng)計(jì)囊胚率。結(jié)果發(fā)現(xiàn),對(duì)照組和試驗(yàn)組的卵裂率(77.68%和81.37%)和囊胚率(17.89%和19.27%)差異不顯著,試驗(yàn)組的極體率(78.03%)顯著高于對(duì)照組(64.55%)。2褪黑激素對(duì)豬卵母細(xì)胞體外發(fā)育過(guò)程中GPx4和Caspase-3表達(dá)的影響通過(guò)RT-PCR方法鑒定外源性褪黑激素是否影響豬體外胚胎的GPx4和Caspase-3基因在轉(zhuǎn)錄水平的表達(dá),探尋影響豬體外胚胎發(fā)育的機(jī)理。豬卵母細(xì)胞體外成熟及孤雌激活后培養(yǎng)基中均添加褪黑激素,收集體外成熟16h、24h、46h的豬卵母細(xì)胞以及2-cell、4-cell和囊胚期的孤雌胚胎,RT-PCR方法鑒定以上基因在轉(zhuǎn)錄水平的表達(dá)情況。褪黑激素能顯著上調(diào)豬卵母細(xì)胞體外培養(yǎng)中胚胎培養(yǎng)階段抗凋亡基因GPx4的表達(dá)(P0.05),抑制培養(yǎng)全程中促凋亡基因Caspase-3的表達(dá)(P0.05)。
[Abstract]:Melatonin (MT) is an indole hormone, which is secreted mainly by pineal gland in human and mammalian body. Other organs and cells also secrete a small amount. During the day, less. MT can enter the organelle through cell membrane and various membrane structures to play the role of antioxidation. At present, the antioxidation and anti-apoptotic effects of MT have been reported in mouse, sheep, chicken and other animal oocytes in vitro maturation and early embryos, but in pig oocytes in vitro maturation is less reported. The effects of melatonin on maturation and embryo development of oocytes in vitro were studied in pigs. The effects of melatonin on the development of porcine oocytes in vitro. (1) the effects of melatonin on the maturation of porcine oocytes in vitro. In vitro maturation medium supplemented with melatonin (10-11 MN 10-9 M 10-7 M 10-7 M 10-5 M 10-3 M) for 46 h, the polar body efflux rate of oocytes in each group was calculated. The results showed that the polar body rates in 10-9M group (77.54%) and 10-7M group (69.2%) were significantly higher than those in control group (67.81%) and other experimental groups. (2) the effect of melatonin on the development of parthenogenetic embryos of porcine oocytes. (2) the effect of melatonin on the development of parthenogenetic embryos of pig oocytes. (2) the eligible oocytes were cultured in maturation medium without melatonin for 46 hours, then parthenogenetic activation was performed and randomly assigned The oocytes were cultured in 6 groups of embryos supplemented with melatonin (10 ~ (-9) M ~ (-1) M ~ (-10 ~ (-9) M ~ (-7) ~ (-7) M ~ (-10 ~ (-7) M ~ (-5) M ~ (-3) M). The cleavage rate was calculated on the 2nd day and blastocyst rate on the 7th day. The results showed that melatonin did not increase the cleavage rate of parthenogenetic oocytes. The blastocyst rate of 10-SM group (20.31%) was significantly higher than that of control group (9.45%) and 10-3M group (10.68%). (_ 3). Oocytes were cultured in vitro for 46 hours and the cleavage rate was counted. The blastocyst rate and blastocyst rate were calculated on the 2nd day and the 7th day after parthenogenetic activation. The results showed that there was no significant difference in cleavage rate (77.68%) and blastocyst rate (17.89% and 19.27%) between control group and experimental group. Effects of melatonin on the expression of GPx4 and Caspase-3 in Porcine oocytes during Development in Vitro, the effects of exogenous melatonin on the expression of GPx4 and Caspase-3 genes in Porcine embryos in Vitro by RT-PCR At the transcriptional level, To explore the mechanism of affecting the development of porcine embryos in vitro. In vitro maturation and parthenogenetic activation of porcine oocytes, melatonin was added to the culture medium. Porcine oocytes were collected for 16 h, 24 h or 46 h after maturation, and 2-cell-4-cell and blastocyst stage parthenogenetic embryos were detected by RT-PCR to identify the expression of the above genes at the transcriptional level. Melatonin could significantly up-regulate the expression of anti-apoptotic gene GPx4 in porcine oocytes in vitro (P0.05) and inhibit the expression of pro-apoptotic gene Caspase-3 during the whole process of culture (P0.05).
【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S852.23

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本文編號(hào)：2078167