小鼠PGCs的分離鑒定及體外培養(yǎng)初步研究
發(fā)布時(shí)間：2019-01-19 13:39
【摘要】：原始生殖細(xì)胞(Primordial germ cells, PGCs)是哺乳動(dòng)物生殖細(xì)胞的前體細(xì)胞,是在胚胎發(fā)育早期具有多能性和獨(dú)特遷移活性的細(xì)胞,近年來已成為研究細(xì)胞命運(yùn)決定、細(xì)胞遷移和表觀遺傳重編程調(diào)節(jié)機(jī)制的重要模型。但由于PGCs細(xì)胞數(shù)量有限、分離純化難度大,這也使得相關(guān)研究還有待深化。本研究對(duì)小鼠PGCs細(xì)胞進(jìn)行了初步的分離、鑒定和體外培養(yǎng)。本研究選取10.5dpc和13.5dpc的小鼠胎兒,分別利用機(jī)械法和酶消化法獲得分散的混合細(xì)胞,對(duì)細(xì)胞進(jìn)行特異性SSEA-1抗體標(biāo)記后,使用流式細(xì)胞儀分選PGCs,并對(duì)分選獲得的細(xì)胞進(jìn)行了堿性磷酸酶(alkaline phosphatase, AKP)染色和多能性標(biāo)記分子OCT4免疫熒光染色鑒定,最后通過RT-PCR技術(shù)檢測(cè)了PGCs特異性基因的表達(dá)。結(jié)果顯示,分選所得細(xì)胞堿性磷酸酶陽(yáng)性率為86.80%,免疫熒光染色表明所分選細(xì)胞呈SSEA-1和OCT4為陽(yáng)性。RT-PCR技術(shù)檢測(cè)結(jié)果顯示分選細(xì)胞表達(dá)Prdml、Lhcgr、Dppa3、Nr6al等PGCs特異性標(biāo)記基因,表明流式細(xì)胞分選獲得的細(xì)胞主要為PGCs。為建立PGCs細(xì)胞的培養(yǎng)方法,本研究采用胚胎生殖細(xì)胞(embryonic germ cells, EG)培養(yǎng)液對(duì)經(jīng)機(jī)械法和酶消化法獲得的細(xì)胞進(jìn)行初步培養(yǎng)并比較了不同飼養(yǎng)層(MEF和STO)和基底膜基質(zhì)(Basement Membrane Matrix)的培養(yǎng)效果。結(jié)果表明,在以上三種培養(yǎng)體系中,13.5dpc胚胎來源的PGCs通過體外培養(yǎng),均未形成集落。10.5dpc胚胎來源的PGCs培養(yǎng)結(jié)果顯示,利用Matrix培養(yǎng)PGCs時(shí)更容易形成集落,其形態(tài)較大呈巢狀,與周圍細(xì)胞存在明顯界限,且堿性磷酸酶染色呈陽(yáng)性,免疫熒光檢測(cè)也表明其表達(dá)多能性O(shè)CT4；而MEF和STO培養(yǎng)體系培養(yǎng)的PGCs,雖然都表達(dá)OCT4,但其形成的集落明顯少于和小于Matrix上形成的集落,且界限不明顯。證明Matrix在PGCs的體外培養(yǎng)中有助于其多能性的維持。綜上所述,本研究利用流式細(xì)胞分選技術(shù)獲得了表達(dá)特異性標(biāo)記分子的小鼠PGCs,利用Matrix進(jìn)行細(xì)胞培養(yǎng)后可形成表達(dá)PGCs特異分子的集落,研究結(jié)果為進(jìn)一步開展對(duì)PGCs的研究奠定了基礎(chǔ)。
[Abstract]:Primordial germ cell (Primordial germ cells, PGCs) is the precursor of mammalian germ cell, which has pluripotent and unique migration activity at the early stage of embryonic development. Important models for regulating mechanisms of cell migration and epigenetic reprogramming. However, due to the limited number of PGCs cells and the difficulty of isolation and purification, the related research needs to be deepened. In this study, mouse PGCs cells were isolated, identified and cultured in vitro. In this study, the mouse fetuses of 10.5dpc and 13.5dpc were selected. The dispersed mixed cells were obtained by mechanical method and enzyme digestion method respectively. The cells were labeled with specific SSEA-1 antibody, and PGCs, was sorted by flow cytometry. The selected cells were identified by alkaline phosphatase (alkaline phosphatase, AKP) staining and multipotent marker OCT4 immunofluorescence staining. Finally, the expression of PGCs specific gene was detected by RT-PCR technique. The results showed that the positive rate of alkaline phosphatase was 86.80%. Immunofluorescence staining showed that the selected cells were positive for SSEA-1 and OCT4. RT-PCR technique showed that the selected cells expressed Prdml,Lhcgr,Dppa3,. Nr6al and other PGCs specific marker genes showed that the cells isolated by flow cytometry were mainly PGCs.. In order to establish the culture method of PGCs cells, (embryonic germ cells, of embryonic germ cells was used in this study. The culture effects of different feeder layers (MEF and STO) and basement membrane matrix (Basement Membrane Matrix) on cells obtained by mechanical and enzymatic digestion were compared. The results showed that the PGCs derived from 13.5dpc embryos did not form colonies in all of the above three culture systems. The results of PGCs culture from 10.5dpc embryos showed that it was easier to form colonies when PGCs was cultured with Matrix. Its morphology is nesting and has obvious boundary with the surrounding cells, and the alkaline phosphatase staining is positive. The immunofluorescence detection also shows that it expresses pluripotent OCT4;. PGCs, in both MEF and STO culture systems expressed OCT4, but the colony formation was less and less than that on Matrix, and the boundary was not obvious. It is proved that Matrix is helpful to maintain the pluripotency of PGCs in vitro. To sum up, the mouse PGCs, expressing specific marker molecules was obtained by flow cytometry, and the colony expressing PGCs specific molecules was formed after Matrix was used for cell culture. The results lay a foundation for further research on PGCs.
【學(xué)位授予單位】：內(nèi)蒙古大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S814
本文編號(hào)：2411422
[Abstract]:Primordial germ cell (Primordial germ cells, PGCs) is the precursor of mammalian germ cell, which has pluripotent and unique migration activity at the early stage of embryonic development. Important models for regulating mechanisms of cell migration and epigenetic reprogramming. However, due to the limited number of PGCs cells and the difficulty of isolation and purification, the related research needs to be deepened. In this study, mouse PGCs cells were isolated, identified and cultured in vitro. In this study, the mouse fetuses of 10.5dpc and 13.5dpc were selected. The dispersed mixed cells were obtained by mechanical method and enzyme digestion method respectively. The cells were labeled with specific SSEA-1 antibody, and PGCs, was sorted by flow cytometry. The selected cells were identified by alkaline phosphatase (alkaline phosphatase, AKP) staining and multipotent marker OCT4 immunofluorescence staining. Finally, the expression of PGCs specific gene was detected by RT-PCR technique. The results showed that the positive rate of alkaline phosphatase was 86.80%. Immunofluorescence staining showed that the selected cells were positive for SSEA-1 and OCT4. RT-PCR technique showed that the selected cells expressed Prdml,Lhcgr,Dppa3,. Nr6al and other PGCs specific marker genes showed that the cells isolated by flow cytometry were mainly PGCs.. In order to establish the culture method of PGCs cells, (embryonic germ cells, of embryonic germ cells was used in this study. The culture effects of different feeder layers (MEF and STO) and basement membrane matrix (Basement Membrane Matrix) on cells obtained by mechanical and enzymatic digestion were compared. The results showed that the PGCs derived from 13.5dpc embryos did not form colonies in all of the above three culture systems. The results of PGCs culture from 10.5dpc embryos showed that it was easier to form colonies when PGCs was cultured with Matrix. Its morphology is nesting and has obvious boundary with the surrounding cells, and the alkaline phosphatase staining is positive. The immunofluorescence detection also shows that it expresses pluripotent OCT4;. PGCs, in both MEF and STO culture systems expressed OCT4, but the colony formation was less and less than that on Matrix, and the boundary was not obvious. It is proved that Matrix is helpful to maintain the pluripotency of PGCs in vitro. To sum up, the mouse PGCs, expressing specific marker molecules was obtained by flow cytometry, and the colony expressing PGCs specific molecules was formed after Matrix was used for cell culture. The results lay a foundation for further research on PGCs.
【學(xué)位授予單位】：內(nèi)蒙古大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S814
【共引文獻(xiàn)】
相關(guān)期刊論文 前3條
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相關(guān)碩士學(xué)位論文 前4條
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2 楊炎;中華鋸齒米蝦性腺早期分化及卵黃蛋白發(fā)生免疫定位[D];河北大學(xué);2010年
3 張立濤;斑馬魚早期性腺發(fā)育及甲基睪酮對(duì)其性分化的影響[D];河北大學(xué);2010年
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，本文編號(hào)：2411422
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