原始生殖细胞抽提物诱导NIH3T3细胞重编程的研究
首发时间:2011-05-06
摘要:目的:探讨原始生殖细胞(primordial germ cells,PGCs)抽提物对小鼠NIH3T3细胞重编程的影响。方法:分离、培养小鼠PGCs;用液氮反复冻融方法制备小鼠PGCs的抽提物;利用抽提物孵育链球菌溶血素O(Streptolysin-O,SLO)打孔后的NIH3T3细胞,分别利用免疫荧光染色和Real time RT-PCR方法检测抽提物处理后的NIH3T3细胞多能性基因和印记基因的表达。结果:建立了小鼠PGCs,碱性磷酸酶检测阳性;Real time RT-PCR结果显示,PGCs抽提物处理1w,NIH3T3细胞开始表达Nanog,Oct4在处理2w后表达,4w后两种基因表达显著升高;印记基因H19 和Igf2 的表达下调;Lamin A的表达轻微下调;免疫荧光结果显示PGCs抽提物处理后NIH3T3细胞表达OCT4。结论:利用原始生殖细胞抽提物孵育体细胞,可使已分化的体细胞发生部分重编程,诱发多潜能基因的表达上调,印记基因和体细胞特定基因的表达下调。
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Reprogramming of NIH3T3 cells in primordial germ cell extracts
Abstract:Objective: To explore the effect of reprogramming on mouse NIH3T3 cells inducing by the cell-extract of primordial germ cells (PGCs). Methods: Isolating and culturing the mouse primordial germ cells. Generation of the cell-extract of PGCs by freezing and thawing repeatedly in liquid nitrogen. Immunofluorescence and real time RT-PCR methods were used to assess the expression of pluripotent genes and imprinted genes in NIH3T3 cells incubated in the extracts. Results: Mouse PGCs were estabalished, which showed positive for alkaline phosphatase staining. The immunofluorescence demonstrated that somatic cells treated with primordial germ cells extracts were positive for OCT4. Real time RT-PCR result showed that somatic cells treated with primordial germ cells extracts began to express Nanog and Oct4 after culture for 1 week and 2 weeks, respectively. Expression of Nanog and Oct4 was up-regulated dramatically 4 weeks later. In contrast, expression of imprinted genes were down-regulated after culture for 1 weeks. In the meanwhile somatic cells-specific gene which named LaminA was down-regulated. Conclusion: Differentiated somatic cells can be reprogrammed partly in vitro by incubated with primordial germ cells extracts, which induced up-regulation of pluripotent genes and down-regulation of imprinted genes and somatic cells-specific genes.
Keywords: primordial germ cells cell extract reprogramming
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