真核表达BMP4成熟肽及其对iPS细胞的分化抑制作用(英文)  

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

作　　者：丁道芳[1] 王臻[2] 徐浩[1] 徐乐勤[1] 梁倩倩[1] 赵永见[1] 施杞[1] 王拥军[1] 

机构地区：[1]上海中医药大学龙华医院脊柱病研究所,上海200032 [2]复旦大学肿瘤医院中心实验室,上海200433

出　　处：《南方医科大学学报》2012年第10期1383-1388,共6页Journal of Southern Medical University

基　　金：Supported by National Basic Research Program of China(973Program,2010CB530400);Key Project of National Natural Science Foundation of China(30930111);Changjiang Scholar Chair Professor Project([2009]17);Shanghai Education Innovation Project(08YZ56);"Shu Guang"Project supported by Shanghai Municipal Education Commission and Shanghai Education Development Foundation(10GG20);Shanghai University Innovation Team Programmer(Shanghai Education Commission,Division 6[2009]);the China Postdoctoral Science Foundation(20090450725)~~

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。Objective To investigate the role of bone morphogenetic protein 4（BMP4） in culturing induced pluripotent stem cells（iPSCs） and the related signal pathways.Methods We amplified the mature peptide of BMP4 from the placenta through RT-PCR,and IgK secretion peptide was ligated to the N-terminal of BMP4 mature peptide.The recombinant plasmid pPYCAG-IgK-BMP4 was transfected into 293T cells and screened with puromycin,and the positive clones for expressing BMP4 were 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 the 3 germ layers and expression levels of pluripotency-associated genes were investigated.Results Smad1 was phosphorylated by BMP4 from the culture medium.iPSCs cultured in the medium supplemented with LIF plus the supernatant containing BMP4 for 3 passages 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 the 3 germ layers.Conclusion BMP4 can be efficiently expressed in mammalian cells to maintain the multipotent differentiation capacity of the iPSCs in in vitro culture.

关 键 词：Igk-BMP4 免疫球蛋白κ链 过表达 诱导型干细胞 细胞分化 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]