Hu Zhiyi, Zhang Ning, Yin Guoyong
Department of Orthopaedics, the First Affiliated Hospital of Nanjing Medical University, Nanjing Jiangsu, 210029, PR China.
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2007 Nov;21(11):1179-83.
To study the function of platelet-derived growth factor (PDGF) in inducing phosphorylation extracellular signal-regulated kinase 1/2 (pERK1/2) localization in osteoblasts.
Primary osteoblasts were isolated and cultured from cranial bone of 10 mice at the age of 3 days, weighting 6-9 g without limitation in male and female. The sixth passage osteoblasts were incubated in 1% serum for 12 hours and divided into 2 groups: treated with DMSO (control group) or with PP2 (experimental group) for 30 minutes. Each group was further divided into 2 subgroups according to with or without PDGF (20 ng/ml) stimulation for 10 minutes. pERK1/2 localization was analysized by immunofluorescence staining in osteoblasts pretreated with or without Src inhibitor PP2. The sixth passage osteoblasts were divided into 2 groups treated with DMSO (control group) or with PP2 (experimental group) for 30 minutes. Each group was further divided into two subgroups according to with or without PDGF (20 ng/ml) stimulation for 10 mintues. The ability of osteoblast migration was determined by wound healing assay. The sixth passage osteoblasts were divided into 2 groups treated with DMSO (control group) or 10 micromol/L PP2 (experimental group) for 30 mintues. Each group was further divided into 2 subgroups according to with or without PDGF (20 ng/ml) stimulation. The pERK1/2 was determined by Western blot in osteoblastic cytoskeleton induced by PDGF.
Immunofluorescence staining showed pERK1/2 localization in osteoblastic nuclears and focal adhesions after PDGF stimulation. PP2 significantly inhibited ERK1/2 localization in focal adhesions, but not in nuclears. The wound healing assay results showed that PP2 significantly inhibited osteoblast migration induced by PDGF. The result of Western blot demonstrated that pERK1/2 in osteoblastic cytoskeleton was significantly inhibited.
Src activation is required for pERK1/2 translocalization to focal adhesions and osteoblasts migration.
研究血小板衍生生长因子（PDGF）在诱导成骨细胞中磷酸化细胞外信号调节激酶1/2（pERK1/2）定位中的作用。
从10只3日龄、体重6 - 9 g的小鼠颅骨中分离培养原代成骨细胞，雌雄不限。将第6代成骨细胞在1%血清中孵育12小时，分为2组：用二甲基亚砜（DMSO）处理（对照组）或用PP2处理（实验组）30分钟。每组再根据有无PDGF（20 ng/ml）刺激10分钟分为2个亚组。在用或不用Src抑制剂PP2预处理的成骨细胞中，通过免疫荧光染色分析pERK1/2的定位。将第6代成骨细胞分为2组，用DMSO处理（对照组）或用PP2（实验组）处理30分钟。每组再根据有无PDGF（20 ng/ml）刺激10分钟分为两个亚组。通过伤口愈合试验测定成骨细胞的迁移能力。将第6代成骨细胞分为2组，用DMSO处理（对照组）或10 μmol/L PP2（实验组）处理30分钟。每组再根据有无PDGF（20 ng/ml）刺激分为2个亚组。通过蛋白质免疫印迹法测定PDGF诱导的成骨细胞骨架中的pERK1/2。
免疫荧光染色显示PDGF刺激后pERK1/2定位于成骨细胞核和黏着斑。PP2显著抑制ERK1/2在黏着斑中的定位，但不影响其在细胞核中的定位。伤口愈合试验结果表明，PP2显著抑制PDGF诱导的成骨细胞迁移。蛋白质免疫印迹法结果显示，成骨细胞骨架中的pERK1/2受到显著抑制。
Src激活是pERK1/2转位至黏着斑和成骨细胞迁移所必需的。
