Liu Ye, Tang Mei Kuen, Cai Dong Qing, Li Ming, Wong Wan Man, Chow Pak Ham, Lee Kenneth K H
Department of Anatomy, Chinese University of Hong Kong, Shatin, Hong Kong.
Proteomics. 2007 Jan;7(1):23-32. doi: 10.1002/pmic.200600456.
In this study, we have used Ki-67 and MF20 mAb to determine how extensively cardiomyocytes proliferate in the postnatal mouse heart. It was established that the cardiomyocytes divided rapidly in 2-day-old hearts. However, at 13 days, the majority of cardiomyocytes had entered into terminal growth arrest and differentiation. We exploited this finding in order to identify proteins that were associated with cardiomyocyte growth and differentiation. The protein profiles of 2- and 13-day-old hearts were established by two-dimensional electrophoresis and compared. Seventeen protein spots were found to be differentially expressed at day 13. Eight of them were up-regulated while the remaining nine protein spots were down-regulated. We focused our attention on 2 of the proteins identified by MALDI-TOF MS, cyclin I and p53, because they are both believed to be involved in cell cycle regulation. Western blot analysis confirmed that both proteins were positively up-regulated in the 13-day-old postnatal heart. To determine directly whether these proteins were associated with cell proliferation, we examined their expression patterns in H9c2 cardiomyocytes maintained in vitro. We established that cyclin I expression was low during the growing phase of H9c2 culture and high during the growth arrest/differentiation phases. In contrast, p53 expression was unchanged during both phases. The various growth phases were confirmed by the presence of cyclin A and growth arrest-specific 1 proteins. We investigated whether silencing cyclin I expression using cyclin I-siRNA could promote an increase in H9c2 cell proliferation. It was determined that silencing cyclin I could enhance a small, but significant, increase in H9c2 cell division. Similar results were obtained for cardiomyocytes extracted from 13-day-old hearts. These results imply that the reason why cardiomyocytes in 13-day-old hearts increased cyclin I expression was probably associated with terminal growth arrest. However, the increase in p53 expression was probably associated with cardiomyocyte differentiation, rather than growth arrest.
在本研究中，我们使用Ki-67和MF20单克隆抗体来确定出生后小鼠心脏中心肌细胞的增殖程度。已证实心肌细胞在2日龄心脏中快速分裂。然而，在13日龄时，大多数心肌细胞已进入终末生长停滞和分化阶段。我们利用这一发现来鉴定与心肌细胞生长和分化相关的蛋白质。通过二维电泳建立了2日龄和13日龄心脏的蛋白质图谱并进行比较。发现17个蛋白点在13日龄时有差异表达。其中8个上调，其余9个蛋白点下调。我们将注意力集中在通过基质辅助激光解吸电离飞行时间质谱鉴定出的2种蛋白质，即细胞周期蛋白I和p53上，因为它们都被认为参与细胞周期调控。蛋白质印迹分析证实这两种蛋白质在出生后13日龄的心脏中均呈阳性上调。为了直接确定这些蛋白质是否与细胞增殖相关，我们检测了它们在体外培养的H9c2心肌细胞中的表达模式。我们发现细胞周期蛋白I在H9c2培养的生长阶段表达低，而在生长停滞/分化阶段表达高。相反，p53在两个阶段的表达均未改变。通过细胞周期蛋白A和生长停滞特异性1蛋白的存在证实了不同的生长阶段。我们研究了使用细胞周期蛋白I小干扰RNA沉默细胞周期蛋白I表达是否能促进H9c2细胞增殖增加。结果表明，沉默细胞周期蛋白I可增强H9c2细胞分裂的小幅但显著增加。从13日龄心脏中提取的心肌细胞也得到了类似结果。这些结果表明，13日龄心脏中心肌细胞细胞周期蛋白I表达增加的原因可能与终末生长停滞有关。然而，p53表达的增加可能与心肌细胞分化有关，而不是生长停滞。
