Expression of BMP4 mature peptide in eukaryotic cells and its differentiation-inhibiting effect in culturing induced pluripotent stem cells

摘要/Abstract

摘要： 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Western blot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。

Abstract: Objective To investigate the role of bone morphogenetic protein4(BMP4) in culturing induced pluripotent stem
cells (iPSCs) and the related signal pathways.MethodsWe amplified the mature peptide of BMP4from the placenta through
RT-PCR, and IgK secretion peptide was ligated to the N-terminal of BMP4 mature peptide. The recombinant plasmid
pPYCAG-IgK-BMP4was transfected into293T cells and screened with puromycin, and the positive clones for expressing
BMP4were verified by cell immunofluorescence and Western blotting. To test the bioactivity of BMP4, iPSCs were cultured in
the medium supplemented with leukemia inhibitory factor (LIF) plus the supernatant containing BMP4, and the cell
phenotype, cell differentiation capacity into lineages of the3germ layers and expression levels of pluripotency-associated
genes were investigated.ResultsSmad1was phosphorylated by BMP4from the culture medium. iPSCs cultured in the
medium supplemented with LIFplus the supernatant containing BMP4for3passages maintained the phenotype of stem cells
with the expression levels of pluripotency-associated genes not affected. These iPSCs also maintained the capacity to
differentiate into cell lineages of the3germ layers.ConclusionBMP4can be efficiently expressed in mammalian cells to
maintain the multipotent differentiation capacity of the iPSCs inin vitroculture.

丁道芳，王臻，徐浩，徐乐勤，梁倩倩，赵永见，施杞，王拥军. Expression of BMP4 mature peptide in eukaryotic cells and its differentiation-inhibiting effect in culturing induced pluripotent stem cells[J]. 南方医科大学学报, 2012, 32(10): 1383-.

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