本文選題：雞胚盤細(xì)胞 + 維甲酸　； 參考：《南京農(nóng)業(yè)大學(xué)》2016年博士論文

【摘要】：憑借獨特的生理和繁殖特性,轉(zhuǎn)基因雞作為生物反應(yīng)器具有重大的價值。雖然近年來轉(zhuǎn)基因雞生產(chǎn)技術(shù)不斷發(fā)展,但制作基因定點編輯的轉(zhuǎn)基因雞仍然非常困難。利用多功能細(xì)胞作為媒介生產(chǎn)基因定點編輯的轉(zhuǎn)基因雞被認(rèn)為是目前最有希望的途徑。雞胚盤細(xì)胞是一種分離于EGK第X期雞胚胚盤明區(qū)的多功能細(xì)胞,它具有數(shù)量龐大、易于分離、易于進(jìn)行基因操作的優(yōu)點。然而,雞胚盤細(xì)胞在移植到受體雞胚后生殖系轉(zhuǎn)化能力極低,因此使用雞胚盤細(xì)胞制作轉(zhuǎn)基因雞難以獲得令人滿意的生產(chǎn)效率。近年來,多篇報道表明使用雞原始生殖細(xì)胞生產(chǎn)轉(zhuǎn)基因雞具有很高的生產(chǎn)效率,但雞原始生殖細(xì)胞在雞胚中含量極少而且很難進(jìn)行分離純化和體外培養(yǎng),因此在體外誘導(dǎo)雞胚盤細(xì)胞向生殖細(xì)胞分化便成了一種值得考慮的提高轉(zhuǎn)基因雞生產(chǎn)效率的途徑。在本研究中,我們使用E8培養(yǎng)基建立了無血清無滋養(yǎng)層的雞胚盤細(xì)胞培養(yǎng)體系;證明了維甲酸(retinoic acid, RA)可以提高雞胚盤細(xì)胞中生殖細(xì)胞相關(guān)基因的表達(dá)并進(jìn)一步研究了相關(guān)的機(jī)制。此外,我們也對多梳蛋白抑制復(fù)合體1 (Polycomb repressive complex 1, PRC1)在雞生殖細(xì)胞減數(shù)分裂起始中的調(diào)控機(jī)制進(jìn)行了研究,該機(jī)制的闡明將為多功能細(xì)胞向生殖細(xì)胞的誘導(dǎo)分化研究提供有價值的參考依據(jù)。1.無血清無滋養(yǎng)層的雞胚盤細(xì)胞培養(yǎng)體系的建立E8培養(yǎng)基是一種在DMEM/F-12培養(yǎng)基中額外添加了 7種成分的干細(xì)胞培養(yǎng)基,試驗證明雞胚盤細(xì)胞可以在無血清無滋養(yǎng)層的E8培養(yǎng)基中培養(yǎng)并呈現(xiàn)出良好的單層貼壁狀態(tài)。通過SSEA-1免疫熒光染色和類胚體形成試驗,我們發(fā)現(xiàn)處于這種狀態(tài)的雞胚盤具有良好的多功能性,而雞胚盤細(xì)胞可以在E8培養(yǎng)基中維持這種單層貼壁狀態(tài)大約兩周的時間。利用上述培養(yǎng)體系,試驗測試了生長因子LIF和SCF的促生長作用,結(jié)果顯示LIF可以顯著提高雞胚盤細(xì)胞的增殖活性和細(xì)胞數(shù)量,但SCF沒有這樣的作用;而且LIF可以顯著提高雞胚盤細(xì)胞的多功能因子PouV和Nanog的表達(dá)。此外,試驗還測試了 PRC1泛素化作用抑制劑PRT4165對雞胚盤細(xì)胞的生長發(fā)育的影響,結(jié)果顯示PRT4165降低了雞胚盤細(xì)胞的增殖活性,通過qRT-PCR檢測證實這種增殖活性的降低是PRT4165抑制雞胚盤細(xì)胞多功能基因的表達(dá)并且增加其凋亡基因的表達(dá)造成的。2. RA通過促進(jìn)Smad1/5磷酸化誘導(dǎo)雞胚盤細(xì)胞向生殖細(xì)胞分化該試驗使用RA對處在單層貼壁狀態(tài)時的雞胚盤細(xì)胞進(jìn)行了誘導(dǎo),結(jié)果顯示RA使雞胚盤細(xì)胞中生殖細(xì)胞相關(guān)基因Stra8的表達(dá)迅速提高了 100倍以上,Dazl和Cvh提高了 4到6倍。結(jié)果也顯示RA使能夠促進(jìn)生殖細(xì)胞轉(zhuǎn)化的基因Bmp2,Bmp4和Bmp8b的表達(dá)顯著提高。上述結(jié)果證明了 RA可以使雞胚盤細(xì)胞向生殖細(xì)胞轉(zhuǎn)化。此外,試驗檢測到RA能在添加后3小時迅速促進(jìn)Smad1/5磷酸化,并且在添加后的6小時使其磷酸化水平達(dá)到峰值。Smad1/5的磷酸化抑制劑dorsomorphin的添加阻止了RA引起的Stra8,Dazl和Cvh表達(dá)的增加。而Smad1/5的磷酸化激活劑SB431542則進(jìn)一步提升了 RA對三個生殖細(xì)胞相關(guān)基因的促表達(dá)作用,而這種SB431542與RA的協(xié)同作用可以被dorsomorphin所抵消;但是缺乏RA的條件下單獨使用SB431542不能提高Stra8,Dazl和Cvh的表達(dá)。通過上述結(jié)果我們推測RA誘導(dǎo)雞胚盤細(xì)胞生殖細(xì)胞轉(zhuǎn)化的機(jī)制是RA首先提高Smad1/5磷酸化,然后磷酸化的Smad1/5作為共激活因子與RA共同促進(jìn)生殖細(xì)胞相關(guān)基因的表達(dá)。RNAi試驗進(jìn)一步確認(rèn)了 Smad1/5在RA誘導(dǎo)雞胚盤細(xì)胞過程中的作用。最后,雞胚盤細(xì)胞移植試驗證明,經(jīng)過誘導(dǎo)的雞胚盤細(xì)胞具有向雞胚性腺遷移的能力。3. PRC1抑制雞胚卵巢中的生殖細(xì)胞減數(shù)分裂起始生殖細(xì)胞減數(shù)分裂起始是生殖細(xì)胞發(fā)育過程中的重要事件,它決定了生殖細(xì)胞的分化方向,也是多功能細(xì)胞在體外向成熟生殖細(xì)胞誘導(dǎo)分化必須經(jīng)歷的過程。本研究中,我們使用雞胚卵巢組織培養(yǎng)并結(jié)合體內(nèi)試驗的方法來探究PRC1在雞卵巢生殖細(xì)胞減數(shù)分裂起始調(diào)控中的作用。研究結(jié)果顯示PRC1核心組份RNF2蛋白在E10.5,E12.5和E14.5的雞胚性腺體細(xì)胞和生殖細(xì)胞的細(xì)胞核中均有表達(dá);Rnf2的mRNA表達(dá)量在E13.5開始上升。在RA存在的前提下,PRT4165可以顯著提高減數(shù)分裂Marker基因Stra8,Scp3和Dmcl的表達(dá),而抑制RA合成的藥物WIN18446則可以阻止PRT4165對三個基因表達(dá)的提升。上述結(jié)果說明PRC1可以抑制雞胚卵巢生殖細(xì)胞的減數(shù)分裂起始,而這個過程具有RA依賴的性質(zhì)。在蛋白水平上,PRT4165可以提高雞胚卵巢中SCP3的表達(dá),而與RA共同處理則可以進(jìn)一步提高SCP3的豐度;免疫熒光分析結(jié)果顯示PRT4165可以提高雞胚卵巢皮質(zhì)中SCP3陽性細(xì)胞的數(shù)量。靶向Rnf2的RANi試驗再次確認(rèn)了 PRC1對雞胚生殖細(xì)胞減數(shù)分裂起始的抑制作用。qRT-PCR試驗并結(jié)合ChIP檢測發(fā)現(xiàn)PRC1可以直接抑制Staa8啟動子對RA的敏感性、間接地維持多功能基因表達(dá)、直接抑制Notch配體表達(dá);通過這三條途徑的協(xié)同作用,PRC1實現(xiàn)了對雞胚卵巢生殖細(xì)胞減數(shù)分裂起始的抑制調(diào)控。RANi試驗確定了 Notch配體Jad1和Dll1的敲減可以抑制減數(shù)分裂相關(guān)基因的表達(dá)。最后,向雞胚羊膜內(nèi)注射PRT4165的體內(nèi)試驗表明PRT4165造成了雞胚卵巢中生殖細(xì)胞減數(shù)分裂起始的提前,并且PRT4165明顯提高了雞胚卵巢皮質(zhì)中的細(xì)胞凋亡水平。綜上所述,我們的研究證明了 E8培養(yǎng)體系能夠在一定時期內(nèi)維持雞胚盤細(xì)胞的正常生長,揭示了 RA促進(jìn)Smad1/5磷酸化并與之一起誘導(dǎo)雞胚盤向生殖細(xì)胞轉(zhuǎn)化。這些結(jié)果不僅進(jìn)一步揭示了 RA在細(xì)胞發(fā)育中的作用機(jī)制,而且為提升雞胚盤細(xì)胞在轉(zhuǎn)基因雞生產(chǎn)中的應(yīng)用價值奠定了基礎(chǔ)。此外,我們的研究證明了 PRC1可以調(diào)控三條相互協(xié)同的途徑來抑制雞胚性腺生殖細(xì)胞減數(shù)分裂的起始;這三條調(diào)控途徑既存在于生殖細(xì)胞也存在于卵巢體細(xì)胞中,這擴(kuò)展了目前對PRC1調(diào)控機(jī)制的理解,也為將來更好地進(jìn)行體外多功能細(xì)胞向生殖細(xì)胞誘導(dǎo)分化的研究提供了一些參考。
[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.
【學(xué)位授予單位】：南京農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：S831

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