Wen Jianyan, Xia Qing, Lu Cailing, Yin Lina, Hu Juan, Gong Yanhua, Yin Bin, Monzen Koshiro, Yuan Jiangang, Qiang Boqin, Zhang Xuemin, Peng Xiaozhong
National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, National Human Genome Center, Beijing 100005, China.
J Cell Biochem. 2007 Sep 1;102(1):149-60. doi: 10.1002/jcb.21285.
A clonal derivative named P19CL6 has been isolated from pluripotent P19 mouse embryonic carcinoma cells, and this subline efficiently differentiates into beating cardiomyocytes when treated with 1% dimethyl sulfoxide (DMSO). It offers a valuable model to study cardiomyocytes differentiation in vitro. In this study, comparative proteomic analysis was used to characterize the protein profiles associated with the DMSO-induced cardiomyocytes differentiation of P19CL6 cells. We demonstrated that P19CL6 cells indeed differentiated into cardiomyocytes after DMSO inducement as they expressed sarcomeric myosin heavy chain (MHC) as well as three cardiac-specific transcription factors (Csx/Nkx-2.5, GATA-4, and MEF2C). Image analysis of silver-stained two-dimensional gels was used to find protein spots that exhibited an at least 1.5-fold change in abundance after successful differentiation. Seventeen protein spots were selected for further analysis by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF-MS) and/or nano-electrospray ionization MS/MS (ESI-MS/MS), and 16 protein spots were identified. The identified proteins are involved in different cellular functions such as metabolism, signal transduction, and cellular organization. To confirm the expression changes of the identified proteins during differentiation, the mRNA levels of six identified proteins (including seven protein spots) were assessed by the real-time polymerase chain reaction and three showed a correlation between mRNA level and protein abundance. As an initial step toward identifying proteins involved in maintaining the differentiated state of cardiomyocytes derived from P19CL6 cells, our data provide some helpful information that may lead to a better understanding of the molecular mechanisms by which P19CL6 cells differentiate into cardiomyocytes after treatment with DMSO.
一种名为P19CL6的克隆衍生物已从多能性P19小鼠胚胎癌细胞中分离出来，当用1%二甲基亚砜（DMSO）处理时，该亚系可有效分化为跳动的心肌细胞。它为体外研究心肌细胞分化提供了一个有价值的模型。在本研究中，采用比较蛋白质组学分析来表征与DMSO诱导的P19CL6细胞心肌细胞分化相关的蛋白质谱。我们证明，DMSO诱导后P19CL6细胞确实分化为心肌细胞，因为它们表达肌节肌球蛋白重链（MHC）以及三种心脏特异性转录因子（Csx/Nkx-2.5、GATA-4和MEF2C）。通过对银染二维凝胶的图像分析来寻找成功分化后丰度变化至少1.5倍的蛋白质斑点。选择了17个蛋白质斑点通过基质辅助激光解吸/电离质谱（MALDI-TOF-MS）和/或纳升电喷雾电离串联质谱（ESI-MS/MS）进行进一步分析，鉴定出16个蛋白质斑点。鉴定出的蛋白质参与不同的细胞功能，如代谢、信号转导和细胞组织。为了确认鉴定出的蛋白质在分化过程中的表达变化，通过实时聚合酶链反应评估了六个鉴定出的蛋白质（包括七个蛋白质斑点）的mRNA水平，其中三个显示mRNA水平与蛋白质丰度之间存在相关性。作为鉴定参与维持源自P19CL6细胞的心肌细胞分化状态的蛋白质的第一步，我们的数据提供了一些有用的信息，可能有助于更好地理解P19CL6细胞经DMSO处理后分化为心肌细胞的分子机制。
