LPA对猪孤雌胚胎早期发育及多能性标志基因的影响

发布时间：2018-03-30 07:41

本文选题：LPA　切入点：囊胚　出处：《广西大学》2017年硕士论文

【摘要】：猪作为我国的大家畜,对我国的农业经济发展起着重要的作用。目前来说获得猪多能干细胞的主要方法是通过体外分离和重编程,但是存在的主要问题都是没有获得“naive”状态的猪ES细胞。为了进一步促进猪胚胎早期发育,获得更利于分离培养获得naive多能性状态的胚胎干细胞(ES)的囊胚材料,我们在本实验探讨Lysophosphatidic acid(LPA)对猪孤雌胚胎早期发育以及相关多能性基因表达的影响并对其信号通路进行了初步探讨。以期为下一步分离获得猪ES获得良好的材料。研究结果总结如下:1.LPA(Lysophosphatidic acid)对猪孤雌胚胎早期发育以及相关胚层的影响。首先对LPA作用的浓度进行探索,从囊赔率、卵裂率以及囊胚细胞数来看,发现50μM的LPA处理浓度要显著优于其他处理组,并且囊胚质量也优于对照组。为了进一步确认前面的试验结果,我们从蛋白水平选用了 OCT4抗体进行了免疫荧光染色,结果显示,50μM的LPA处理浓度免疫荧光强度也优于其他实验组和对照组,所以确定了 50μM的LPA处理浓度进行了后续的实验。随后探索了LPA对原始内胚层、原始外胚层以及滋养层标志基因的影响。QRT-PCR结果显示:LPA对原始外胚层标志基因没有显著影响,但是LPA可以显著上调滋养层标志基因的表达并且可以极显著地下调原始内胚层标志基因的表达。以上结果表明,LPA可以促进早期猪孤雌囊胚的发育,并且对猪早期胚胎原始内胚层有着重要的影响。2.LPA作用于猪孤雌胚胎相关信号通路的探索。首先探索FGF信号通路对猪孤雌胚胎原始内胚层的影响,QRT-PCR结果显示:30ng/mL的bFGF可以显著上调猪囊胚原始内胚层GATT4基因。在同时添加LPA和bFGF后,猪囊胚原始内胚层标志基因GATA4和单独添加LPA时结果一致,即GATA4基因显著下降。随后探索LPA是否通过ROCK信号通路起作用。使用ROCK信号通路抑制剂Y27632抑制ROCK信号通路表达以后再添加LPA后,猪囊胚原始内胚层标志基因GATA4没有显著变化。以上结果表明,与小鼠早期胚胎一样,FGF信号通路任然可以调节猪的原始内胚层的发育,而LPA主要通过ROCK信号通路对猪原始内胚层起作用,并且作用强度可能大于FGF信号通路。3.为了更进一步的研究LPA对猪早期囊胚发育中多能性基因表达的影响,对“naive”和“primed”两类候选基因进行了定量测定,结果显示:LPA对“naive”态基因没有显著影响,但是能显著下调“primed”多能基因NODAL和Activin-A的表达,并且能显著提高ES细胞自我更新因子OCT4和c-Myc的表达。以上结果表明:LPA可以促进猪孤雌胚胎早期发育,并且可能通过抑制primed多能基因的表达来促进和维持“naive”状态细胞的多能性,以上的研究结果有望促进naive猪ES细胞系的分离培养及建系。
[Abstract]:Pigs, as the large livestock of our country, play an important role in the development of our agricultural economy. At present, the main methods of obtaining porcine pluripotent stem cells are isolation and reprogramming in vitro. In order to further promote the early development of porcine embryos, the blastocyst materials for isolation and culture of naive pluripotent embryonic stem cells were obtained. In this study, we investigated the effect of Lysophosphatidic acididae on the early development of parthenogenetic embryos and the expression of related pluripotent genes, and studied its signal pathway in order to obtain a good material for the further isolation of porcine es. The results are summarized as follows: 1. The effects of LPA Lysophosphatidic acid) on the early development of porcine parthenogenetic embryos and related embryo layers. From the sac odds, cleavage rate and blastocyst cell count, we found that 50 渭 M LPA treatment concentration was significantly higher than other treatment groups, and the blastocyst quality was also superior to the control group. We selected OCT4 antibody from protein level for immunofluorescence staining. The results showed that the immunofluorescence intensity of 50 渭 M LPA treatment was better than that of other experimental groups and control groups. So the concentration of 50 渭 M LPA was determined to carry on the subsequent experiment. Then we explored the effect of LPA on the primitive endoderm, primitive ectoderm and trophoblast marker genes. The results of QRT-PCR showed that: LPA had no significant effect on the primitive ectodermal marker gene. However, LPA could significantly up-regulate the expression of trophoblast marker gene and down-regulate the expression of primitive endoderm marker gene. LPA-induced signal pathway related to parthenogenetic embryos was explored. Firstly, the effects of FGF signaling pathway on the primitive endoderm of parthenogenetic embryos were explored. The results of QRT-PCR showed that: 30 ng / mL bFGF. The primordial endoderm GATT4 gene of porcine blastocyst could be upregulated significantly. After the addition of LPA and bFGF, The results of the primordial endoderm marker gene GATA4 of pig blastocyst were consistent with those of LPA alone. The expression of ROCK signal pathway was inhibited by Y27632, and the expression of ROCK signal pathway was inhibited by Y27632. After LPA was added, the expression of ROCK signal pathway was inhibited by Y27632. There was no significant change in the primordial endoderm marker gene GATA4 in pig blastocysts. However, LPA mainly acts on the primitive endoderm of pigs through ROCK signaling pathway, and its action intensity may be greater than that of FGF signaling pathway .3.In order to further study the effect of LPA on the expression of pluripotent genes in early blastocyst development, The quantitative analysis of the candidate genes of "naive" and "primed" showed that there was no significant effect on the gene expression of "naive", but the expression of NODAL and Activin-A of the "primed" pluripotent gene could be significantly down-regulated. These results suggest that OCT4 can promote the early development of porcine parthenogenetic embryos, and it may promote and maintain the pluripotency of "naive" state cells by inhibiting the expression of primed pluripotent genes. The above results are expected to promote the isolation, culture and establishment of naive porcine es cell lines.
【学位授予单位】：广西大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S828

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