microRNA-145和microRNA-483对牛核移植胚胎早期发育的影响

发布时间：2018-03-04 16:04

本文选题：牛　切入点：体细胞核移植　出处：《西北农林科技大学》2016年博士论文　论文类型：学位论文

【摘要】：利用体细胞核移植技术(SCNT)来生产克隆动物和转基因动物被认为是最为有效的方法,特别是在动物遗传育种,动物品种保护,生物医药等诸多方面该方法具有巨大的应用前景。但是目前SCNT动物的生产效率还较低,通常认为主要原因是由于在体细胞核移植过程中体细胞的核不完全重编程所导致的,因此研究如何提高SCNT胚胎的重编程效率,特别是提高植入前胚胎的发育率和胚胎质量,进而提高SCNT动物的生产效率,对于SCNT技术的广泛推广应用具有十分重要的意义。本试验通过对比体外培养条件下的牛SCNT胚胎与体外受精(IVF)胚胎在植入前的早期发育过程中发育率的差异,检测在发育率具有显著差异的时期两类胚胎中与重编程密切相关基因的表达差异,并通过microRNAs的RNA干扰技术来提高牛SCNT胚胎植入前胚胎发育率和胚胎质量,主要研究结果如下:1.对比牛SCNT胚胎与IVF胚胎在体外培养环境下在植入前五个发育节点胚胎发育率的异常,发现在2-cell和4-cell时期两类胚胎的发育率没有显著差异,而SCNT胚胎在8-cell、桑葚胚和囊胚时期的发育率要显著低于IVF胚胎的发育率。进一步检测在8-cell、桑葚胚和囊胚这三个时期牛SCNT胚胎与IVF胚胎中与重编程密切相关的基因OCT4、SOX2、KLF4,以及IGFBP3和IGF2的表达量的差异,发现牛SCNT胚胎中OCT4、SOX2、KLF4这三个基因的表达量要显著低于IVF胚胎中的表达量,提示这与牛SCNT胚胎发育率的异常有关;同样发现相对于牛IVF胚胎,SCNT胚胎中IGFBP3的表达量显著减少,而IGF2的表达量显著增加,提示这与牛胚胎发育率异常相关,并且与克隆动物过度生长有关。2.由于microRNAs能够特异性的抑制其靶基因的表达,再利用microRNAs inhibitor能够特异性的抑制具体microRNA作用的这一特点,通过抑制microRNA-145的表达来促进其靶基因OCT4、SOX2、KLF4的表达,发现在用40μM的microRNA-145 inhibitor处理牛SCNT胚胎后,胚胎在8-cell、桑葚胚和囊胚这三个时期的发育率有了显著提高。进一步检测后发现胚胎中OCT4、SOX2、KLF4的表达量有了显著的升高,说明microRNA-145 inhibitor能够提高胚胎中OCT4、SOX2、KLF4基因的表达,提高克隆胚的重编程效率。3.IGFBP3能够与IGF2特异性结合从而抑制IGF2发挥生物学作用,同时IGFBP3也是microRNA-483的靶基因,在试验中我们发现在用100μM的microRNA-483 inhibitor处理牛SCNT胚胎后,胚胎在8-cell、桑葚胚和囊胚这三个时期的发育率有了显著提高。进一步检测后发现胚胎中IGFBP3的表达量有了显著升高,并且IGF2的表达量有了显著降低,说明microRNA-483 inhibitor能够提高胚胎中IGFBP3的表达量,增加的IGFBP3能够结合更多的IGF2,从而降低IGF2的表达量,纠正因重编程异常所引起的胚胎发育异常。
[Abstract]:The use of somatic cell nuclear transfer (SCNT) technology to produce cloned and transgenic animals is considered to be the most effective method, especially in animal genetics and breeding, animal variety protection, This method has great application prospect in many aspects, such as biomedicine. But at present, the production efficiency of SCNT animals is still low. It is generally believed that the main reason is the incomplete nuclear reprogramming of somatic cells during somatic cell nuclear transfer. Therefore, how to improve the efficiency of reprogramming of SCNT embryos, especially the development rate and embryo quality of preimplantation embryos, so as to improve the productivity of SCNT animals is studied. It is of great significance for the wide application of SCNT technology. In this study, we compared the developmental rates of bovine SCNT embryos in vitro with those of IVF embryos in vitro during their early development before implantation. The expression of genes closely related to reprogramming was detected in two types of embryos with significant differences in development rate, and the RNA interference technique of microRNAs was used to improve the development rate and embryo quality of bovine SCNT embryos before implantation. The main results were as follows: 1. There was no significant difference in the development rates of bovine SCNT embryos and IVF embryos in the first five developmental nodes during 2-cell and 4-cell periods, compared with those of IVF embryos in vitro. The development rate of SCNT embryos at 8-cell, morula and blastocyst stages was significantly lower than that of IVF embryos. Further, the gene OCT4SOX2 and KLF4, which was closely related to reprogramming in bovine SCNT embryos and IVF embryos in 8-cell, morula and blastocyst stages, was further detected. And the differences in the expression levels of IGFBP3 and IGF2, It was found that the expression of OCT4, SOX2, KLF4 in bovine SCNT embryos was significantly lower than that in IVF embryos, which suggested that the expression of OCT4SOX2KLF4 gene was related to the abnormal development rate of bovine SCNT embryos, and it was also found that the expression of IGFBP3 in bovine IVF embryos was significantly lower than that in bovine IVF embryos. However, the expression of IGF2 was significantly increased, which suggested that it was related to the abnormal rate of bovine embryo development and to the over-growth of cloned animals. Because microRNAs could specifically inhibit the expression of its target gene, Using the characteristic that microRNAs inhibitor can specifically inhibit the effect of specific microRNA, and by inhibiting the expression of microRNA-145, we can promote the expression of its target gene OCT4, SOX2, KLF4. It was found that after the treatment of bovine SCNT embryos with 40 渭 M microRNA-145 inhibitor, The development rate of embryos in 8-cell, morula and blastocyst was significantly increased. Further detection showed that the expression of OCT4OX2SOX2KLF4 was significantly increased in embryos, which indicated that microRNA-145 inhibitor could increase the expression of OCT4SOX2KLF4 gene in embryos. 3. IGFBP3 can specifically bind with IGF2 to inhibit the biological effect of IGF2, and IGFBP3 is also a target gene of microRNA-483. In the experiment, we found that after 100 渭 M microRNA-483 inhibitor was used to treat bovine SCNT embryos, The developmental rate of embryos in 8-cell, morula and blastocyst increased significantly. The expression of IGFBP3 and IGF2 in embryos were significantly increased, and the expression of IGF2 was significantly decreased, after further detection, there was a significant increase in the expression of IGFBP3 in embryos, and a significant decrease in the expression of IGF2 in embryos. The results showed that microRNA-483 inhibitor could increase the expression of IGFBP3 in embryos, and the increased IGFBP3 could combine with more IGF2, thus reducing the expression of IGF2 and correcting the abnormal development of embryos caused by abnormal reprogramming.
【学位授予单位】：西北农林科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S814.8

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