本文選題：AZD5438　切入點(diǎn)：豬　出處：《延邊大學(xué)》2017年碩士論文

【摘要】：現(xiàn)如今,豬卵母細(xì)胞激活方法和相關(guān)激活參數(shù)的研究己經(jīng)比較成熟,但激活的效率仍然很低。此外,由于卵母細(xì)胞激活是細(xì)胞核移植等生物技術(shù)的關(guān)鍵環(huán)節(jié),提高孤雌激活效率也可以促進(jìn)細(xì)胞核移植技術(shù)的提高。AZD5438是一種細(xì)胞周期蛋白依賴性激酶(cyclin-dependent kinase,CDK)抑制劑,在體外試驗(yàn)中,AZD5438可通過抑制CDK底物的磷酸化,從而阻斷細(xì)胞周期。本試驗(yàn)?zāi)康氖翘剿鰽ZD5438對(duì)豬孤雌激活與體細(xì)胞核移植胚胎體外發(fā)育的影響,為研究新型的MPF抑制劑,優(yōu)化豬卵母細(xì)胞體外激活程序奠定基礎(chǔ)。本研究主要內(nèi)容及結(jié)果如下:1.試驗(yàn)探索了 AZD5438的最佳處理?xiàng)l件。采用不同處理濃度(10μM,20μM和50μM)的AZD5438分別處理電激活后的豬卵母細(xì)胞孤雌激活(Parthenogenetic Activation,PA)胚胎,通過比較體外豬PA囊胚率得出,添加10μM AZD5438的試驗(yàn)組囊胚率最高,并且顯著高于5 μg/ml細(xì)胞松弛素 B(cytochalasin B,CB)組(46.4%vs.34.5%,P0.05)。隨后,采用10μM AZD5438分別處理電激活后的豬PA胚胎不同時(shí)間(2h,4h和6h)。結(jié)果表明,在10μMAZD5438處理4 h的條件下,豬PA囊胚發(fā)育率高于其他時(shí)間組(42.8%vs.38.6%,37.2%),但并無顯著差異性。2.試驗(yàn)將 AZD5438 和 6-二甲基氨基嘌呤(6-dimethylaminopurine,6-DMAP)對(duì)豬 PA 胚胎與體細(xì)胞核移植(Somatic Cell Nuclear Transfer,SCNT)胚胎早期發(fā)育的影響進(jìn)行比較。采用10μMAZD5438和2mM6-DMAP分別處理電激活后的豬PA胚胎和豬SCNT胚胎4h,比較PA囊胚率和豬SCNT囊胚率。結(jié)果表明,10μM AZD5438處理4 h的條件下得到了與6-DMAP處理組相似的豬PA囊胚率(42.4%vs.34.3%)和SCNT囊胚率(13.40%vs.11.11%)。3.試驗(yàn)對(duì)經(jīng)10μMAZD5438處理4h后得到的豬PA囊胚進(jìn)行核型分析。結(jié)果表明,10μM AZD5438組豬囊胚中60%為二倍體,10%豬PA囊胚的為單倍體,10%豬PA囊胚的為四倍體,20%豬PA囊胚的為雜合體。4.試驗(yàn)將使用10μM AZD5438和5 μg/ml CB處理4h得到的豬PA胚胎內(nèi)成熟促進(jìn)因子(MPF)活性水平進(jìn)行測定。結(jié)果表明,10μMAZD5438處理顯著降低豬PA胚胎中MPF活性水平(125.8 vs.288.7,P0.05)。5.試驗(yàn)采用Real-time PCR方法檢測多能相關(guān)基因(Sox2、0ct4和Nanog)和凋亡相關(guān)基因(Bcl-2和Bax)的相對(duì)表達(dá)量,研究11μ AZD5438處理豬孤雌激活胚胎4 h后,對(duì)基因表達(dá)、胚胎發(fā)育能力的影響。結(jié)果表明,10 μM AZD5438處理豬PA胚胎4h,對(duì)多能相關(guān)基因(Sox2、0ct4和Nanog)和凋亡相關(guān)基因(Bcl-2和Bax)的相對(duì)表達(dá)水平與CB對(duì)照組(5μg/mml)無顯著差異。上述研究結(jié)果表明,AZD5438通過降低豬PA胚胎內(nèi)MPF活性水平來提高豬PA胚胎體外發(fā)育能力,并在10μM AZD5438處理4h的條件下,能夠提高豬孤雌激活胚胎與體細(xì)胞核移植胚胎體外發(fā)育水平。除此之外,孤雌囊胚中多能性相關(guān)基因(Sox2、Oct4和Nanog)和凋亡相關(guān)基因(Bax和Bcl-2)的相對(duì)mRNA表達(dá)水平?jīng)]有差異。證明AZD5438處理能夠提高孤雌胚的體外發(fā)育水平,可作為一種新型MPF抑制劑應(yīng)用于卵母細(xì)胞激活體系之中。
[Abstract]:Nowadays, porcine oocytes activation methods and related activation parameters have been well studied, but the efficiency of activation is still very low.In addition, as oocyte activation is a key link in biological techniques such as nuclear transplantation, increasing the efficiency of parthenogenetic activation can also promote the enhancement of nuclear transplantation techniques. AZD5438 is a cyclin dependent kinase inhibitor, CDKK.In vitro, AZD5438 could block cell cycle by inhibiting phosphorylation of CDK substrate.The aim of this study was to explore the effects of AZD5438 on the in vitro development of porcine parthenogenetic and somatic nuclear transfer embryos, and to lay a foundation for the study of novel MPF inhibitors and the optimization of porcine oocyte activation in vitro.The main contents and results of this study are as follows: 1.The optimum treatment conditions of AZD5438 were investigated.The parthenogenetic activation (PAA) embryos of porcine oocytes were treated with AZD5438 with different concentrations of 10 渭 M and 50 渭 M respectively. By comparing the blastocyst rate of porcine PA in vitro, the highest blastocyst rate was found in the experimental group supplemented with 10 渭 M AZD5438.And it was significantly higher than that of 5 渭 g/ml cytochalasin B(cytochalasin group (46.4vs.34.5).Then, 10 渭 M AZD5438 was used to treat the electrically activated porcine PA embryos for 4 h and 6 h, respectively.The results showed that under the condition of 10 渭 MAZD5438 for 4 h, the blastocyst development rate of porcine PA was higher than that of other time groups (42.8 vs.38.6%), but there was no significant difference.The effects of AZD5438 and 6-dimethylaminopurine 6-DMAPon on the early embryo development of porcine PA embryos and somatic Cell Nuclear transfer NTs were compared.Porcine PA embryos and porcine SCNT embryos were treated with 10 渭 MAZD5438 and 2mM6-DMAP for 4 h, respectively. The blastocyst rates of PA and SCNT were compared.The results showed that the blastocyst rate of PA was similar to that of 6-DMAP treated with 10 渭 M AZD5438 for 4 h. The blastocyst rate and SCNT blastocyst rate were 42.4vs.34.3and 13.40 vs 11.111.3.The karyotype of porcine PA blastocysts treated with 10 渭 MAZD5438 for 4 h was analyzed.The results showed that 60% of porcine blastocysts in 10 渭 M AZD5438 group were diploid 10% porcine PA blastocysts and 10% haploid 10% porcine PA blastocysts were tetraploid or 20% porcine PA blastocysts were heterozygous.The activity of porcine PA embryo maturation promoting factor (MPF), which was treated with 10 渭 M AZD5438 and 5 渭 g/ml CB for 4 h, was determined.The results showed that 10 渭 MAZD5438 treatment significantly reduced the level of MPF activity in porcine PA embryos.Real-time PCR method was used to detect the relative expression of Sox2Ct4 and Nanog4 and apoptosis-related genes Bcl 2 and Bax. the effects of 11 渭 AZD5438 treatment on gene expression and embryonic development were studied after 4 h of porcine parthenogenetic activation.The results showed that there was no significant difference in the relative expression levels of multifunctional genes (Sox2Ct4 and Nanog4) and apoptosis-related genes (Bcl-2 and Bax) between 10 渭 M AZD5438 and 5 渭 g / mmlCB control group for 4 h.The results showed that AZD5438 could improve the ability of porcine PA embryos to develop in vitro by reducing the level of MPF activity in PA embryos, and could improve the level of development of porcine parthenogenetic activated embryos and somatic nuclear transfer embryos after 4 h treatment with 10 渭 M AZD5438.In addition, there was no difference in the relative mRNA expression levels of polymorphic genes such as Sox2Oct4 and Nanog4 and apoptosis-related genes (Bax and Bcl-2) in parthenogenetic blastocysts.The results showed that AZD5438 treatment could improve the development of parthenogenetic embryos in vitro and could be used as a new type of MPF inhibitor in oocyte activation system.
【學(xué)位授予單位】：延邊大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S828

【參考文獻(xiàn)】

相關(guān)期刊論文 前6條

1 向舒;楊金姬;石金月;張順;王建;陸鳳花;石德順;;兔卵母細(xì)胞孤雌激活方法的研究[J];中國獸醫(yī)學(xué)報(bào);2011年07期

2 田見暉,劉國世,曾申明,朱士恩,蔡元,劉煥營,張忠誠,吳常信;電脈沖激活對(duì)體外成熟豬卵母細(xì)胞卵裂率和囊胚率的影響[J];農(nóng)業(yè)生物技術(shù)學(xué)報(bào);2004年04期

3 廉莉,廉穎,吳昱琪,徐營,朱子玉,雷蕾,陳大元;兔卵母細(xì)胞的孤雌激活[J];中國獸醫(yī)科技;2003年04期

4 張果平,黃永宏,李峰;哺乳動(dòng)物卵母細(xì)胞孤雌激活及孤雌胚發(fā)育研究進(jìn)展[J];內(nèi)蒙古畜牧科學(xué);2003年01期

5 蘭國成,王子玉,馬所峰,苗義良,譚景和;乙醇及6-DMAP對(duì)小鼠卵母細(xì)胞孤雌激活的研究[J];細(xì)胞生物學(xué)雜志;2002年05期

6 李光鵬,孟慶剛,魏鵬,李子義,孫興參,譚景和;亞胺環(huán)己酮對(duì)豬卵母細(xì)胞人工孤雌激活作用的研究[J];畜牧獸醫(yī)學(xué)報(bào);2001年05期

相關(guān)碩士學(xué)位論文 前1條

1 康雅雅;TSA和5-Aza-Cdr對(duì)豬孤雌激活胚胎發(fā)育及相關(guān)基因表達(dá)影響的研究[D];東北農(nóng)業(yè)大學(xué);2013年

，

本文編號(hào)：1709906