本文關鍵詞： 原始生殖細胞 轉基因 移植 遷移　出處：《廣西大學》2016年碩士論文　論文類型：學位論文

【摘要】：原始生殖細胞(Primodial germ cells,PGCs)作為配子的前體細胞,負責將遺傳信息傳遞給下一代。將分離的PGCs經(jīng)過體外培養(yǎng)擴增以及轉基因修飾后,移植到受體胚內(nèi)能產(chǎn)生生殖系嵌合體,這是生產(chǎn)轉基因禽類的一個理想途徑。但在實際的研究操作中,由于對PGCs遷移和歸巢性腺知識的缺乏,轉基因嵌合體的制備方法沒有優(yōu)化,成功效率非常低。本研究旨在探索PGCs在注射到受體胚胎后的遷移機制以及影響其遷移效率的因素,為提高轉基因雞的生產(chǎn)效率提供一定的依據(jù)。首先,我們從廣西黃羽雞(yellow feather chicken,YF chicken) 13-15期雞胚血液中分離PGCs進行體外培養(yǎng)擴增,經(jīng)轉基因修飾后,對獲得的能穩(wěn)定遺傳的GFP-PGCs進行鑒定。結果發(fā)現(xiàn),經(jīng)過體外培養(yǎng)及轉基因修飾的PGCs, SSEA-1染色呈陽性,RT-PCR分析結果顯示這些PGCs表達了Dazl、Cvh、PouV、Cdh、Nanog基因,說明了我們獲得的GFP-PGCs細胞株仍保留其生物學特性。隨后,我們將雞胚隨機分成4組,分別孵育50h、55h、60h、65h后移植GFP-PGCs,探索受體胚齡對PGCs遷移的影響。結果發(fā)現(xiàn),受體雞胚孵育50h移植PGCs的遷移效率較孵育55h、60h、65h組高,孵育時間越長移植PGCs遷移效率越低。在探究移植細胞數(shù)對PGCs遷移的影響試驗中,雞胚隨機分成四組,孵育50h后分別注射1000、3000、10000、30000個GFP-PGCs,結果顯示,移植的最優(yōu)細胞數(shù)為10000個／胚,移植的PGCs少于10000個／胚時,隨著移植細胞數(shù)的增加,遷移的PGCs增加,當移植的PGCs多于10000個／胚時,遷移的PGCs不再增加。我們將移植10000個PGCs的雞胚孵出,出殼4d小雞睪丸內(nèi)發(fā)現(xiàn)外源GFP-PGCs,證明了外源GFP-PGCs能在受體性腺內(nèi)生長增殖。隨后,成熟雄性嵌合體雞精液DNA PCR結果顯示,4個假定嵌合體中有1個呈GFP陽性。最后,我們探究了CXCR4信號通路在PGCs遷移過程中的作用。RT-PCR結果顯示,PGCs表達了趨化因子受體Cxcr4基因。此外,我們在移植的細胞滴中分別添加CXCR4信號通路抑制劑AMD3100和WZ811, AMD3100濃度為OnM、10nM、50nM, WZ811為OnM、1nM、2nM,試驗結果表明,50nMAMD3100以及1nM、 2nM WZ811能明顯抑制PGCs的遷移(P0.05)。證明了SDF-1/CXCR4信號通路參與調(diào)控PGCs在早期雞胚中的遷移和歸巢。綜上所述,廣西黃羽雞PGCs能在體外長期培養(yǎng),經(jīng)轉基因修飾后仍保留其生物學特性。受體雞胚孵育50h為最佳移植時間,每個雞胚移植10000個PGCs遷移至性腺的PGCs最多,歸巢至性腺的PGCs能在受體睪丸中正常增殖生長。SDF-1/CXCR4信號通路參與介導PGCs的體內(nèi)遷移過程。
[Abstract]:Primodial germ cells (PGCs), the progenitor of gametes, are responsible for transmitting genetic information to the next generation. After in vitro culture, amplification and modification of the isolated PGCs, transplanted into the recipient embryos, the chimerism of the reproductive line can be produced. This is an ideal way to produce transgenic birds, but in practical research operations, due to the lack of knowledge of PGCs migration and homing gonad, the preparation method of transgenic chimera has not been optimized. The purpose of this study was to explore the migration mechanism of PGCs after injection into recipient embryos and the factors affecting its migration efficiency, and to provide a basis for improving the productivity of transgenic chickens. We isolated and amplified PGCs from the blood of chicken embryo of stage 13-15 of Guangxi yellow feather chickenen. After modified by transgenic method, the stable GFP-PGCs was identified. After in vitro culture and transgenic PGCs, SSEA-1 staining positive RT-PCR analysis showed that these PGCs expressed the gene of Dazlus Cvhus PouVu CDHN Nanog, indicating that our obtained GFP-PGCs cell line still retains its biological characteristics. Then, we randomly divided the chicken embryo into 4 groups. The effects of embryo age on PGCs migration were investigated. The results showed that the migration efficiency of PGCs transplanted after 50 h incubation of recipient chicken embryos was higher than that of 55 h incubated chicken embryos at 60 h or 65 h after incubating for 60 h or 65 h, respectively, and the effect of recipient embryo age on PGCs migration was higher than that in recipient chicken embryos incubated for 50 h or 60 h or 65 h. The effect of the number of transplanted cells on PGCs migration was investigated. Chicken embryos were randomly divided into four groups after incubating for 50 hours. After 50 hours of incubation, 30000 GFP-PGCswere injected into 1000 ~ 3000g / g, respectively. The results showed that the optimal number of transplanted cells was 10 000 / embryo. When the transplanted PGCs is less than 10, 000 / embryo, as the number of transplanted cells increases, the migrated PGCs increases, and when the transplanted PGCs is more than 10, 000 / embryo, the migrated PGCs does not increase. We hatch 10, 000 PGCs chicken embryos. Exogenous GFP-PGCswas found in the testis of 4 days out of shell, which proved that exogenous GFP-PGCs could grow and proliferate in the receptor gonads. Subsequently, DNA PCR of mature male chimera chicken semen showed that one of the four hypothetical chimeras was GFP positive. We investigated the role of CXCR4 signaling pathway in PGCs migration. RT-PCR results showed that CXCR4 expressed chemokine receptor Cxcr4 gene. We added CXCR4 signaling pathway inhibitor AMD3100 and WZ811 into the transplanted cells, the concentration of AMD3100 was OnM10 nMU 50nM, and WZ811 was OnM1nMU 2nM. the results showed that P0.05and 50nMAMD3100 and 1nMMAMD3100 significantly inhibited PGCs migration P0.05. it was proved that SDF-1/CXCR4 signaling pathway was involved in the regulation of PGCs in the early stage. Migration and homing in chicken embryos. Guangxi yellow feather chicken PGCs could be cultured for a long time in vitro, and its biological characteristics were retained after transgenic modification. The best transfer time was 50 hours after incubation of recipient chicken embryos, and 10 000 PGCs per chicken embryo transferred to the PGCs of gonad. Homing to gonad PGCs can mediate the migration of PGCs in vivo by normal proliferation and growth. SDF-1 / CXCR4 signaling pathway.
【學位授予單位】：廣西大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：S831

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