BGJ398对猪孤雌胚胎发育及滋养层发育潜能相关基因表达的影响

发布时间：2018-09-11 15:12

【摘要】：胚胎由一个全能的受精卵分裂形成多个发育潜力几乎相同的卵裂球,随着胚胎的形态的不断变化,形成一个空腔,此时,卵裂球随之分化形成内细胞团(Inner cell mass,ICM),后续发育成为胎体和部分胎盘组织,和外层的滋养层细胞(Trophoblast,TE),其后发育成为胎盘部分。TE在胚胎植入和妊娠维持中起重要作用。前人研究表明抑制水牛早期囊胚内FGF信号通路能够提高囊胚整体的细胞数,而且细胞数的增加主要来自滋养层。另外在猪的研究也表明,抑制FGF信号通路可以在不影响胚胎正常发育和相关多能性基因的表达的前提下,显著提高滋养层标志基因CDX2的表达量,然而激活FGF信号通路可以使得CDX2的表达量明显下降。虽然机理暂时不明,但是这有可能成为一个极其有前景的研究方向。目前对猪早期胚胎内滋养层的研究甚少,对于在早期胚胎内滋养层的发育情况,并没有一个衡量其发育潜力的标准。通常都以滋养层标志基因CDX2的表达量作为滋养层细胞发育情况的标准。基于滋养层在胚胎在子宫中的定位、附着、侵入过程中的重要地位,建立一个衡量其发育潜力的标准以及提高其细胞数和相关标志/功能基因的表达,为提高后续的胚胎移植效率提供了一种非常有实际意义的探讨。本研究主要内容如下:对成熟卵细胞进行孤雌激活(Parthenogenetic activation,PA)后,分别添加浓度为5μM、10μM、15μM、20μM的FGF信号通路抑制剂BGJ-398到基础培养基内培养168h收集囊胚,与DMSO对照组对比囊胚率、囊胚内滋养层细胞数和相关基因表达量进行比较。结果发现:1.在基础培养基内添加BGJ398并不影响囊胚的形成和正常发育,但是与DMSO对照组相比,5μM组囊胚率有显著提高(29.64%vs 22.97%,P0.05),10μM组囊胚率有一定增长但是差异并不显著(24.68%vs 22.97%,P0.05)。当浓度增加到15μM、20μM时,囊胚率降低,说明浓度过高对囊胚的形成起到抑制作用。对囊胚进行Hoechst染色细胞手动计数,与对照组相比,5μM组囊胚细胞数有显著提高(54.2±7.92vs45.16±8.75,P0.05),10μM组囊胚细胞数有一定增长但是差异并不显著(45.16±8.75 vs 48.2±4.32,P0.05),15μM、20μM组囊胚平均细胞数均少于对照组。选取最优浓度实验组5μM对囊胚多能性相关基因表达量进行检测。结果表明相对于对照组,处理组的多能性相关基因KLF4的表达量显著提高了 6.28倍(P0.05),而OCT4表达量为对照组的1.50倍(P0.05),差异不显著;原始外胚层标志基因SOX2的表达量显著提高了 3.45倍(P0.05);囊胚内滋养层标志基因CDX2表达量显著提高了 2.93倍(P0.05)。2.BGJ398促进以猪PA囊胚滋养层相关标志基因的表达。通过PCR检测我们发现:GATA3,TEAD4,CCDX2,OCT4,TERT,OEMES,FGFR2在囊胚中均有表达。但是PPARγ,HAND1,MMP2,MMP9以及ELF5,CYP11A1,HSD17B1没有检测到表达。上述结果提示GATA3,TEAD4,CDX2,OCT4,TERT,OEMES,FGFR2可以作为后续实验中滋养层细胞检测的评价基因。选取5μM处理组和有报道过的10μM处理组的囊胚对筛选出的滋养层标志物表达量进行定量分析,研究结果发现,与对照组相比,调控滋养层形成与分化的CDX2的上游基因GATA3表达量在5μM组和10μM组分别提高了 1.49倍和2.14倍(P0.05),但差异不显著;端粒酶活性基因TERT的表达得到了显著提高,与对照组相比分别提高了 3.03倍和1.70倍(P0.05);CDX2上游调控因子TEAD4表达量也得到显著提高,5μM组和10μM组表达量分别为对照组的2.00倍和2.09倍(P0.05);FGFR2由于通路抑制表达量随着抑制剂浓度上升而降低;5μM组和10μM组EOMES表达量分别为对照组的3.18倍(P0.05)和1.52倍(P0.05)。上述实验结果说明BGJ398能够促进滋养层细胞功能相关基因GATA3,TEAD4 CDX2,OCT4,TERT,OEES表达量的显著提高,推测 BJG398能够提高滋养层细胞发育潜力,从而可能间接提高胚胎着床能力。综上所述,在猪胚胎培养基添加5μMBGJ398,能够促进PA囊胚率以及囊胚细胞数,同时促进多能性相关基因以及与囊胚滋养层细胞发育潜力相关标志基因的表达,为下一步探讨提高猪胚胎移植成功率奠定基础。
[Abstract]:The embryo divides from one omnipotent fertilized egg to form several blastomeres with almost the same developmental potential. As the shape of the embryo changes, a cavity is formed. At this time, the blastomere then differentiates into an inner cell mass (ICM), which then develops into the fetus and part of the placental tissue, and the trophoblast (T cell) in the outer layer. E), which later became part of the placenta, plays an important role in embryo implantation and pregnancy maintenance. Previous studies have shown that inhibiting the FGF signaling pathway in Buffalo blastocysts can increase the number of cells in the blastocyst * and the increase in cell numbers mainly comes from trophoblasts. In addition, studies in pigs also show that inhibition of FGF signaling pathway can not affect.TE. On the premise of normal embryo development and expression of related pluripotent genes, the expression level of trophoblast marker gene CDX2 can be significantly improved. However, the activation of FGF signaling pathway can significantly reduce the expression of CDX2. Although the mechanism is temporarily unknown, it may become a very promising research direction. * Few studies have been done on trophoblasts, and there is no criterion for measuring the development potential of trophoblasts in early embryos. The expression of CDX2, a trophoblast marker, is usually used as a criterion for trophoblastic cell development. A criterion for evaluating the development potential and increasing the number of cells and the expression of related markers/functional genes provide a very practical approach to improve the efficiency of subsequent embryo transfer. BGJ-398, a 5,10,15,20 Mu inhibitor of FGF signaling pathway, was cultured in basal medium for 168 hours to collect blastocysts. The blastocyst rate, the number of trophoblast cells and the expression of related genes were compared with DMSO control group. The results showed that: 1. BGJ-398 added to basal medium did not affect the formation and normal development of blastocysts, but with DMSO control group. Compared with the DMSO control group, the blastocyst rate of 5 mu M group increased significantly (29.64% vs 22.97%, P 0.05). The blastocyst rate of 10 mu M group increased slightly but the difference was not significant (24.68% vs 22.97%, P 0.05). When the concentration increased to 15 mu M, 20 mu M, the blastocyst rate decreased, indicating that excessive concentration inhibited the formation of blastocysts. Comparing with the control group, the number of blastocyst cells in 5_ M group was significantly increased (54.2 7.92 vs 45.16 8.75, P 0.05). The number of blastocyst cells in 10_ M group increased slightly but the difference was not significant (45.16 8.75 vs 48.2 4.32, P 0.05). The average number of blastocysts in 15_ M and 20_ M groups was lower than that in the control group. The results showed that compared with the control group, the expression of KLF4 was significantly increased by 6.28 times (P 0.05), while the expression of OCT4 was 1.50 times (P 0.05), and the expression of SOX2 was significantly increased by 3.45 times (P 0.05). The expression level of CDX2 gene was increased by 2.93 * (P0.05).2.BGJ398 significantly to promote the expression of the marker related genes of pig PA blastocyst trophoblast. By PCR detection, we found that GATA3, TEAD4, CCDX2, OCT4, TERT, OEMES, and OCT4 were expressed in blastocysts. These results suggest that GATA3, TEAD4, CDX2, OCT4, TERT, OEMES and FGFR2 can be used as evaluation genes for trophoblast cell detection in subsequent experiments. The expression of GATA3 in CDX2 upstream gene increased by 1.49 times and 2.14 times in 5 and 10 mu M groups respectively (P 0.05), but the difference was not significant; the expression of TERT in CDX2 upstream gene increased by 3.03 and 1.70 times compared with the control group (P 0.05); the expression of TEAD4 in CDX2 upstream regulatory factor also increased significantly, and the expression of TEAD4 in 5 mu M group increased significantly. The expression levels of FGFR2 were 2.00 and 2.09 times (P 0.05) as compared with the control group, and that of EOMES was 3.18 times (P 0.05) and 1.52 times (P 0.05) as compared with the control group. 3, the expression level of TEAD4 CDX2, OCT4, TERT and OEES increased significantly. It was speculated that BJG398 could enhance the developmental potential of trophoblastic cells and thus indirectly enhance the embryo implantation ability. * in conclusion, adding 5 MBGJ398 to pig embryo culture medium can promote the number of blastocysts and blastocyst cells, and promote pluripotent related genes and blastocyst nourishment. The expression of marker genes related to the development of cell * s layer lays the foundation for further discussion on the success rate of porcine embryo transfer.
【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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