AZD5438对猪孤雌激活与体细胞核移植胚胎体外发育的影响

发布时间：2018-04-04 12:13

本文选题：AZD5438　切入点：猪　出处：《延边大学》2017年硕士论文

【摘要】：现如今,猪卵母细胞激活方法和相关激活参数的研究己经比较成熟,但激活的效率仍然很低。此外,由于卵母细胞激活是细胞核移植等生物技术的关键环节,提高孤雌激活效率也可以促进细胞核移植技术的提高。AZD5438是一种细胞周期蛋白依赖性激酶(cyclin-dependent kinase,CDK)抑制剂,在体外试验中,AZD5438可通过抑制CDK底物的磷酸化,从而阻断细胞周期。本试验目的是探索AZD5438对猪孤雌激活与体细胞核移植胚胎体外发育的影响,为研究新型的MPF抑制剂,优化猪卵母细胞体外激活程序奠定基础。本研究主要内容及结果如下:1.试验探索了 AZD5438的最佳处理条件。采用不同处理浓度(10μM,20μM和50μM)的AZD5438分别处理电激活后的猪卵母细胞孤雌激活(Parthenogenetic Activation,PA)胚胎,通过比较体外猪PA囊胚率得出,添加10μM AZD5438的试验组囊胚率最高,并且显著高于5 μg/ml细胞松弛素 B(cytochalasin B,CB)组(46.4%vs.34.5%,P0.05)。随后,采用10μM AZD5438分别处理电激活后的猪PA胚胎不同时间(2h,4h和6h)。结果表明,在10μMAZD5438处理4 h的条件下,猪PA囊胚发育率高于其他时间组(42.8%vs.38.6%,37.2%),但并无显著差异性。2.试验将 AZD5438 和 6-二甲基氨基嘌呤(6-dimethylaminopurine,6-DMAP)对猪 PA 胚胎与体细胞核移植(Somatic Cell Nuclear Transfer,SCNT)胚胎早期发育的影响进行比较。采用10μMAZD5438和2mM6-DMAP分别处理电激活后的猪PA胚胎和猪SCNT胚胎4h,比较PA囊胚率和猪SCNT囊胚率。结果表明,10μM AZD5438处理4 h的条件下得到了与6-DMAP处理组相似的猪PA囊胚率(42.4%vs.34.3%)和SCNT囊胚率(13.40%vs.11.11%)。3.试验对经10μMAZD5438处理4h后得到的猪PA囊胚进行核型分析。结果表明,10μM AZD5438组猪囊胚中60%为二倍体,10%猪PA囊胚的为单倍体,10%猪PA囊胚的为四倍体,20%猪PA囊胚的为杂合体。4.试验将使用10μM AZD5438和5 μg/ml CB处理4h得到的猪PA胚胎内成熟促进因子(MPF)活性水平进行测定。结果表明,10μMAZD5438处理显著降低猪PA胚胎中MPF活性水平(125.8 vs.288.7,P0.05)。5.试验采用Real-time PCR方法检测多能相关基因(Sox2、0ct4和Nanog)和凋亡相关基因(Bcl-2和Bax)的相对表达量,研究11μ AZD5438处理猪孤雌激活胚胎4 h后,对基因表达、胚胎发育能力的影响。结果表明,10 μM AZD5438处理猪PA胚胎4h,对多能相关基因(Sox2、0ct4和Nanog)和凋亡相关基因(Bcl-2和Bax)的相对表达水平与CB对照组(5μg/mml)无显著差异。上述研究结果表明,AZD5438通过降低猪PA胚胎内MPF活性水平来提高猪PA胚胎体外发育能力,并在10μM AZD5438处理4h的条件下,能够提高猪孤雌激活胚胎与体细胞核移植胚胎体外发育水平。除此之外,孤雌囊胚中多能性相关基因(Sox2、Oct4和Nanog)和凋亡相关基因(Bax和Bcl-2)的相对mRNA表达水平没有差异。证明AZD5438处理能够提高孤雌胚的体外发育水平,可作为一种新型MPF抑制剂应用于卵母细胞激活体系之中。
[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.
【学位授予单位】：延边大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S828

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