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OALib Journal期刊
ISSN: 2333-9721
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expressionofbmp4maturepeptideineukaryoticcellsanditsdifferentiation-inhibitingeffectinculturinginducedpluripotentstemcells
Keywords: igk-bmp4,免疫球蛋白κ链,过表达,诱导型干细胞,细胞分化
Abstract:
目的研究bmp4因子在诱导型干细胞（ips细胞）培养过程中的作用和起作用的相关通路。方法通过rt-pcr的方法从小鼠的胎盘组织中扩增bmp4成熟肽，并且在其n末端连接igk分泌肽，此融合的片段克隆至ppycag载体上。重组质粒ppycag-igk-bmp4转染至293t17细胞中并进行嘌呤霉素的筛选。通过细胞免疫荧光和westernblot方法鉴定出表达bmp4的阳性克隆。为进一步检测上清中bmp4的生物活性，ips细胞培养于含bmp4因子的细胞培养上清和lif因子中，连续培养3代，并进一步观察细胞表型、三胚层细胞的分化潜能和多潜能相关基因的表达水平。结果smad1被分泌至上清中的bmp4磷酸化。当培养基中含bmp4同时加入lif因子后，可以有效抑制ips细胞分化。在此种方法培养3代后，不仅ips细胞的表型可以有效维持，ips细胞中多潜能相关的基因表达水平也未受到影响，同时这些ips细胞仍具有向三个胚层分化的能力。结论bmp4可高效表达在真核细胞中，有效地使培养ips细胞保持其多向分化潜能。
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