小鼠PGCs的分离鉴定及体外培养初步研究
发布时间：2019-01-19 13:39
【摘要】：原始生殖细胞(Primordial germ cells, PGCs)是哺乳动物生殖细胞的前体细胞,是在胚胎发育早期具有多能性和独特迁移活性的细胞,近年来已成为研究细胞命运决定、细胞迁移和表观遗传重编程调节机制的重要模型。但由于PGCs细胞数量有限、分离纯化难度大,这也使得相关研究还有待深化。本研究对小鼠PGCs细胞进行了初步的分离、鉴定和体外培养。本研究选取10.5dpc和13.5dpc的小鼠胎儿,分别利用机械法和酶消化法获得分散的混合细胞,对细胞进行特异性SSEA-1抗体标记后,使用流式细胞仪分选PGCs,并对分选获得的细胞进行了碱性磷酸酶(alkaline phosphatase, AKP)染色和多能性标记分子OCT4免疫荧光染色鉴定,最后通过RT-PCR技术检测了PGCs特异性基因的表达。结果显示,分选所得细胞碱性磷酸酶阳性率为86.80%,免疫荧光染色表明所分选细胞呈SSEA-1和OCT4为阳性。RT-PCR技术检测结果显示分选细胞表达Prdml、Lhcgr、Dppa3、Nr6al等PGCs特异性标记基因,表明流式细胞分选获得的细胞主要为PGCs。为建立PGCs细胞的培养方法,本研究采用胚胎生殖细胞(embryonic germ cells, EG)培养液对经机械法和酶消化法获得的细胞进行初步培养并比较了不同饲养层(MEF和STO)和基底膜基质(Basement Membrane Matrix)的培养效果。结果表明,在以上三种培养体系中,13.5dpc胚胎来源的PGCs通过体外培养,均未形成集落。10.5dpc胚胎来源的PGCs培养结果显示,利用Matrix培养PGCs时更容易形成集落,其形态较大呈巢状,与周围细胞存在明显界限,且碱性磷酸酶染色呈阳性,免疫荧光检测也表明其表达多能性OCT4；而MEF和STO培养体系培养的PGCs,虽然都表达OCT4,但其形成的集落明显少于和小于Matrix上形成的集落,且界限不明显。证明Matrix在PGCs的体外培养中有助于其多能性的维持。综上所述,本研究利用流式细胞分选技术获得了表达特异性标记分子的小鼠PGCs,利用Matrix进行细胞培养后可形成表达PGCs特异分子的集落,研究结果为进一步开展对PGCs的研究奠定了基础。
[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.
【学位授予单位】：内蒙古大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S814
本文编号：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.
【学位授予单位】：内蒙古大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S814
【共引文献】
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相关硕士学位论文 前4条
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2 杨炎;中华锯齿米虾性腺早期分化及卵黄蛋白发生免疫定位[D];河北大学;2010年
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4 李慧君;中华鳖性腺分化及温度对其影响[D];河北大学;2012年
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