维甲酸和多梳蛋白抑制复合体1对鸡胚盘细胞诱导分化以及生殖细胞减数分裂起始的调控研究

发布时间：2018-06-23 21:25

本文选题：鸡胚盘细胞 + 维甲酸　；参考：《南京农业大学》2016年博士论文

【摘要】：凭借独特的生理和繁殖特性,转基因鸡作为生物反应器具有重大的价值。虽然近年来转基因鸡生产技术不断发展,但制作基因定点编辑的转基因鸡仍然非常困难。利用多功能细胞作为媒介生产基因定点编辑的转基因鸡被认为是目前最有希望的途径。鸡胚盘细胞是一种分离于EGK第X期鸡胚胚盘明区的多功能细胞,它具有数量庞大、易于分离、易于进行基因操作的优点。然而,鸡胚盘细胞在移植到受体鸡胚后生殖系转化能力极低,因此使用鸡胚盘细胞制作转基因鸡难以获得令人满意的生产效率。近年来,多篇报道表明使用鸡原始生殖细胞生产转基因鸡具有很高的生产效率,但鸡原始生殖细胞在鸡胚中含量极少而且很难进行分离纯化和体外培养,因此在体外诱导鸡胚盘细胞向生殖细胞分化便成了一种值得考虑的提高转基因鸡生产效率的途径。在本研究中,我们使用E8培养基建立了无血清无滋养层的鸡胚盘细胞培养体系;证明了维甲酸(retinoic acid, RA)可以提高鸡胚盘细胞中生殖细胞相关基因的表达并进一步研究了相关的机制。此外,我们也对多梳蛋白抑制复合体1 (Polycomb repressive complex 1, PRC1)在鸡生殖细胞减数分裂起始中的调控机制进行了研究,该机制的阐明将为多功能细胞向生殖细胞的诱导分化研究提供有价值的参考依据。1.无血清无滋养层的鸡胚盘细胞培养体系的建立E8培养基是一种在DMEM/F-12培养基中额外添加了 7种成分的干细胞培养基,试验证明鸡胚盘细胞可以在无血清无滋养层的E8培养基中培养并呈现出良好的单层贴壁状态。通过SSEA-1免疫荧光染色和类胚体形成试验,我们发现处于这种状态的鸡胚盘具有良好的多功能性,而鸡胚盘细胞可以在E8培养基中维持这种单层贴壁状态大约两周的时间。利用上述培养体系,试验测试了生长因子LIF和SCF的促生长作用,结果显示LIF可以显著提高鸡胚盘细胞的增殖活性和细胞数量,但SCF没有这样的作用;而且LIF可以显著提高鸡胚盘细胞的多功能因子PouV和Nanog的表达。此外,试验还测试了 PRC1泛素化作用抑制剂PRT4165对鸡胚盘细胞的生长发育的影响,结果显示PRT4165降低了鸡胚盘细胞的增殖活性,通过qRT-PCR检测证实这种增殖活性的降低是PRT4165抑制鸡胚盘细胞多功能基因的表达并且增加其凋亡基因的表达造成的。2. RA通过促进Smad1/5磷酸化诱导鸡胚盘细胞向生殖细胞分化该试验使用RA对处在单层贴壁状态时的鸡胚盘细胞进行了诱导,结果显示RA使鸡胚盘细胞中生殖细胞相关基因Stra8的表达迅速提高了 100倍以上,Dazl和Cvh提高了 4到6倍。结果也显示RA使能够促进生殖细胞转化的基因Bmp2,Bmp4和Bmp8b的表达显著提高。上述结果证明了 RA可以使鸡胚盘细胞向生殖细胞转化。此外,试验检测到RA能在添加后3小时迅速促进Smad1/5磷酸化,并且在添加后的6小时使其磷酸化水平达到峰值。Smad1/5的磷酸化抑制剂dorsomorphin的添加阻止了RA引起的Stra8,Dazl和Cvh表达的增加。而Smad1/5的磷酸化激活剂SB431542则进一步提升了 RA对三个生殖细胞相关基因的促表达作用,而这种SB431542与RA的协同作用可以被dorsomorphin所抵消;但是缺乏RA的条件下单独使用SB431542不能提高Stra8,Dazl和Cvh的表达。通过上述结果我们推测RA诱导鸡胚盘细胞生殖细胞转化的机制是RA首先提高Smad1/5磷酸化,然后磷酸化的Smad1/5作为共激活因子与RA共同促进生殖细胞相关基因的表达。RNAi试验进一步确认了 Smad1/5在RA诱导鸡胚盘细胞过程中的作用。最后,鸡胚盘细胞移植试验证明,经过诱导的鸡胚盘细胞具有向鸡胚性腺迁移的能力。3. PRC1抑制鸡胚卵巢中的生殖细胞减数分裂起始生殖细胞减数分裂起始是生殖细胞发育过程中的重要事件,它决定了生殖细胞的分化方向,也是多功能细胞在体外向成熟生殖细胞诱导分化必须经历的过程。本研究中,我们使用鸡胚卵巢组织培养并结合体内试验的方法来探究PRC1在鸡卵巢生殖细胞减数分裂起始调控中的作用。研究结果显示PRC1核心组份RNF2蛋白在E10.5,E12.5和E14.5的鸡胚性腺体细胞和生殖细胞的细胞核中均有表达;Rnf2的mRNA表达量在E13.5开始上升。在RA存在的前提下,PRT4165可以显著提高减数分裂Marker基因Stra8,Scp3和Dmcl的表达,而抑制RA合成的药物WIN18446则可以阻止PRT4165对三个基因表达的提升。上述结果说明PRC1可以抑制鸡胚卵巢生殖细胞的减数分裂起始,而这个过程具有RA依赖的性质。在蛋白水平上,PRT4165可以提高鸡胚卵巢中SCP3的表达,而与RA共同处理则可以进一步提高SCP3的丰度;免疫荧光分析结果显示PRT4165可以提高鸡胚卵巢皮质中SCP3阳性细胞的数量。靶向Rnf2的RANi试验再次确认了 PRC1对鸡胚生殖细胞减数分裂起始的抑制作用。qRT-PCR试验并结合ChIP检测发现PRC1可以直接抑制Staa8启动子对RA的敏感性、间接地维持多功能基因表达、直接抑制Notch配体表达;通过这三条途径的协同作用,PRC1实现了对鸡胚卵巢生殖细胞减数分裂起始的抑制调控。RANi试验确定了 Notch配体Jad1和Dll1的敲减可以抑制减数分裂相关基因的表达。最后,向鸡胚羊膜内注射PRT4165的体内试验表明PRT4165造成了鸡胚卵巢中生殖细胞减数分裂起始的提前,并且PRT4165明显提高了鸡胚卵巢皮质中的细胞凋亡水平。综上所述,我们的研究证明了 E8培养体系能够在一定时期内维持鸡胚盘细胞的正常生长,揭示了 RA促进Smad1/5磷酸化并与之一起诱导鸡胚盘向生殖细胞转化。这些结果不仅进一步揭示了 RA在细胞发育中的作用机制,而且为提升鸡胚盘细胞在转基因鸡生产中的应用价值奠定了基础。此外,我们的研究证明了 PRC1可以调控三条相互协同的途径来抑制鸡胚性腺生殖细胞减数分裂的起始;这三条调控途径既存在于生殖细胞也存在于卵巢体细胞中,这扩展了目前对PRC1调控机制的理解,也为将来更好地进行体外多功能细胞向生殖细胞诱导分化的研究提供了一些参考。
[Abstract]:With unique physiological and reproductive characteristics, transgenic chicken is of great value as a bioreactor. Although the production technology of transgenic chicken has been developing continuously in recent years, it is still very difficult to make genetically modified chickens. The chicken embryo disc cell is a kind of multifunctional cell separated from the EGK stage X chicken embryo disc clear area. It has a large number, easy to separate and easy to carry out the advantage of gene operation. However, the transformation ability of the chicken embryo disc cells is very low after transplantation to the recipient chicken embryo, so it is difficult to use chicken embryo disc cells to make transgenic chickens. In recent years, many reports have shown that the use of chicken primordial germ cells to produce genetically modified chickens has a high production efficiency, but the content of chicken primordial germ cells in chicken embryos is very small and difficult to be purified and cultured in vitro. Therefore, the differentiation of chicken embryo disc cells into germ cells is a kind of differentiation in vitro. In this study, we used the E8 medium to establish a serum-free and non trophoblastic chicken embryo disc cell culture system. It is proved that retinoic acid (RA) can improve the expression of germ cell related genes in the chicken embryo disc cells and further study the related mechanisms. We also studied the regulatory mechanism of Polycomb repressive complex 1 (PRC1) in the meiotic initiation of chicken reproductive cells, which will provide a valuable reference for the study of the induction and differentiation of multifunctional cells to reproductive cells by.1. without serum-free serum-free chicken embryo disc cell culture. The establishment of the E8 culture medium is a stem cell culture medium added to the DMEM/F-12 medium with 7 additional components. The experiment proved that the chicken embryo disc cells can be cultured in the E8 medium without serum-free trophoblastic layer and showed a good monolayer adherence. We found that the SSEA-1 immunofluorescence staining and the embryoid body formation test have been found. The chicken embryo disc in this state has good multifunction, and the chicken embryo disc cells can maintain the monolayer adherence for about two weeks in the E8 medium. The growth factor LIF and SCF growth promoting effect was tested by the above culture system. The results showed that LIF could significantly increase the proliferation activity of the chicken oderm cells. The number of cells, but SCF did not play such a role, and LIF could significantly increase the expression of the multifunctional factors PouV and Nanog in the chicken embryo disc cells. In addition, the experiment also tested the effect of PRC1 ubiquitination inhibitor PRT4165 on the growth and development of the chicken oderblast cells. The results showed that PRT4165 reduced the proliferation activity of the chicken embryo disc cells and passed qRT-PCR through qRT-PCR. The detection showed that the decrease of the proliferation activity was PRT4165 inhibiting the expression of the multifunction gene of the chicken embryo disc cells and increasing the expression of the apoptosis gene, and the.2. RA induced the differentiation of the chicken embryo disc cells to the germ cells by promoting Smad1/5 phosphorylation. The RA cells were induced by RA on the monolayer oderblast cells. The results showed that RA increased the expression of germ cell related gene Stra8 in chicken embryo disc cells more than 100 times, and Dazl and Cvh increased by 4 to 6 times. The results also showed that RA could promote the gene Bmp2 of reproductive cell transformation, and the expression of Bmp4 and Bmp8b increased significantly. The results showed that RA could convert the chicken embryo disc cells into the germ cells. In addition, the test detected that RA could rapidly promote the phosphorylation of Smad1/5 3 hours after addition, and the addition of phosphorylation inhibitor dorsomorphin, which reached the peak of phosphorylation level of.Smad1/5 after the addition of 6 hours after addition, prevented RA induced Stra8, Dazl and Cvh expression increased. Smad1/5's phosphorylation activator SB431542 further promoted RA. The synergism of three reproductive cell related genes, and the synergistic effect of this SB431542 and RA can be offset by dorsomorphin; but the lack of RA alone SB431542 can not improve the expression of Stra8, Dazl and Cvh. Through the above results we speculate that the mechanism of RA induced reproductive cell transformation in chicken embryonic disc cells is RA first. High Smad1/5 phosphorylation, then phosphorylated Smad1/5 as co activating factor and RA to promote the expression of germ cell related genes,.RNAi test further confirmed the role of Smad1/5 in the induction of chicken embryo disc cells by RA. Finally, chicken embryo disc cell transplantation test showed that the induced chicken embryo disc cells migrated to the gonads of chicken embryo. The ability of.3. PRC1 to inhibit the meiosis initiation of the meiosis of germ cells in the embryo and ovary of the chicken embryo is an important event in the development of germ cells. It determines the direction of differentiation of the germ cells, and is a process that the multifunctional cells must undergo in vitro differentiation into the mature germ cells. In this study, we use chickens The role of PRC1 in the initiation and regulation of meiosis in the ovarian germ cells of the chicken ovary was studied by the embryo ovarian tissue culture and in vivo test. The results showed that the PRC1 core component RNF2 protein was expressed in the nucleus of the embryogenic glandular and reproductive cells of E10.5, E12.5 and E14.5, and the mRNA expression of Rnf2 began to rise in E13.5. Under the presence of RA, PRT4165 can significantly increase the expression of the meiotic Marker gene Stra8, Scp3 and Dmcl, and the inhibition of RA synthesis, WIN18446, can prevent PRT4165's enhancement of the expression of three genes. The results indicate that PRC1 can inhibit the meiosis initiation of the ovarian germ cells of the chicken embryo, and this process has a RA dependence. On the protein level, PRT4165 can improve the expression of SCP3 in the ovaries of chicken embryo, and the co processing with RA can further improve the abundance of SCP3. The results of immunofluorescence analysis show that PRT4165 can increase the number of SCP3 positive cells in the ovary cortex of chicken embryo. The RANi test of the target Rnf2 reconfirms the number of PRC1 on the germ cell of chicken embryo. The inhibitory effect of.QRT-PCR and ChIP detection found that PRC1 could directly inhibit the sensitivity of Staa8 promoter to RA, indirectly maintain the expression of multifunction gene, and directly inhibit the expression of Notch ligand. Through the synergy of these three pathways, PRC1 has realized the inhibition of the meiosis initiation of the chicken embryo ovarian germ cells. The ANi test confirmed that the knockout of the Notch ligand Jad1 and Dll1 could inhibit the expression of meiosis related genes. Finally, the in vivo test of PRT4165 injection into the chicken embryo amniotic membrane showed that PRT4165 caused the initiation of meiosis in the germ ovary of chicken embryo, and PRT4165 significantly increased the level of cell apoptosis in the chicken embryo ovary cortex. To sum up, our study demonstrated that the E8 culture system can maintain the normal growth of the chicken embryo disc cells for a certain period, and reveal that RA promotes Smad1/5 phosphorylation and induces the transformation of the chicken embryo disc to the germ cells. These results not only further reveal the mechanism of the action of RA in the cell development, but also improve the chicken embryo disc. In addition, our study shows that PRC1 can regulate three synergistic approaches to inhibit the initiation of meiosis in the gonadal germ cells of the chicken embryo; the three regulatory pathways exist both in the germ cells and in the ovarian cells, which extends the current PRC1 regulator. The understanding of the system also provides some references for the further study of the differentiation of multipotent cells into germ cells in the future.
【学位授予单位】：南京农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S831

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