本文選題：LPA　切入點：囊胚　出處：《廣西大學(xué)》2017年碩士論文

【摘要】：豬作為我國的大家畜,對我國的農(nóng)業(yè)經(jīng)濟(jì)發(fā)展起著重要的作用。目前來說獲得豬多能干細(xì)胞的主要方法是通過體外分離和重編程,但是存在的主要問題都是沒有獲得“naive”狀態(tài)的豬ES細(xì)胞。為了進(jìn)一步促進(jìn)豬胚胎早期發(fā)育,獲得更利于分離培養(yǎng)獲得naive多能性狀態(tài)的胚胎干細(xì)胞(ES)的囊胚材料,我們在本實驗探討Lysophosphatidic acid(LPA)對豬孤雌胚胎早期發(fā)育以及相關(guān)多能性基因表達(dá)的影響并對其信號通路進(jìn)行了初步探討。以期為下一步分離獲得豬ES獲得良好的材料。研究結(jié)果總結(jié)如下:1.LPA(Lysophosphatidic acid)對豬孤雌胚胎早期發(fā)育以及相關(guān)胚層的影響。首先對LPA作用的濃度進(jìn)行探索,從囊賠率、卵裂率以及囊胚細(xì)胞數(shù)來看,發(fā)現(xiàn)50μM的LPA處理濃度要顯著優(yōu)于其他處理組,并且囊胚質(zhì)量也優(yōu)于對照組。為了進(jìn)一步確認(rèn)前面的試驗結(jié)果,我們從蛋白水平選用了 OCT4抗體進(jìn)行了免疫熒光染色,結(jié)果顯示,50μM的LPA處理濃度免疫熒光強(qiáng)度也優(yōu)于其他實驗組和對照組,所以確定了 50μM的LPA處理濃度進(jìn)行了后續(xù)的實驗。隨后探索了LPA對原始內(nèi)胚層、原始外胚層以及滋養(yǎng)層標(biāo)志基因的影響。QRT-PCR結(jié)果顯示:LPA對原始外胚層標(biāo)志基因沒有顯著影響,但是LPA可以顯著上調(diào)滋養(yǎng)層標(biāo)志基因的表達(dá)并且可以極顯著地下調(diào)原始內(nèi)胚層標(biāo)志基因的表達(dá)。以上結(jié)果表明,LPA可以促進(jìn)早期豬孤雌囊胚的發(fā)育,并且對豬早期胚胎原始內(nèi)胚層有著重要的影響。2.LPA作用于豬孤雌胚胎相關(guān)信號通路的探索。首先探索FGF信號通路對豬孤雌胚胎原始內(nèi)胚層的影響,QRT-PCR結(jié)果顯示:30ng/mL的bFGF可以顯著上調(diào)豬囊胚原始內(nèi)胚層GATT4基因。在同時添加LPA和bFGF后,豬囊胚原始內(nèi)胚層標(biāo)志基因GATA4和單獨添加LPA時結(jié)果一致,即GATA4基因顯著下降。隨后探索LPA是否通過ROCK信號通路起作用。使用ROCK信號通路抑制劑Y27632抑制ROCK信號通路表達(dá)以后再添加LPA后,豬囊胚原始內(nèi)胚層標(biāo)志基因GATA4沒有顯著變化。以上結(jié)果表明,與小鼠早期胚胎一樣,FGF信號通路任然可以調(diào)節(jié)豬的原始內(nèi)胚層的發(fā)育,而LPA主要通過ROCK信號通路對豬原始內(nèi)胚層起作用,并且作用強(qiáng)度可能大于FGF信號通路。3.為了更進(jìn)一步的研究LPA對豬早期囊胚發(fā)育中多能性基因表達(dá)的影響,對“naive”和“primed”兩類候選基因進(jìn)行了定量測定,結(jié)果顯示:LPA對“naive”態(tài)基因沒有顯著影響,但是能顯著下調(diào)“primed”多能基因NODAL和Activin-A的表達(dá),并且能顯著提高ES細(xì)胞自我更新因子OCT4和c-Myc的表達(dá)。以上結(jié)果表明:LPA可以促進(jìn)豬孤雌胚胎早期發(fā)育,并且可能通過抑制primed多能基因的表達(dá)來促進(jìn)和維持“naive”狀態(tài)細(xì)胞的多能性,以上的研究結(jié)果有望促進(jìn)naive豬ES細(xì)胞系的分離培養(yǎng)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.
【學(xué)位授予單位】：廣西大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S828

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

1 ;Effects of lysophosphatidic acid on human colon cancer cells and its mechanisms of action[J];World Journal of Gastroenterology;2009年36期

2 ;Regulation of cellular adhesion molecule expression in murine oocytes, peri-implantation and post-implantation embryos[J];Cell Research;2002年Z2期

，

本文編號：1684967