摘要
目的探讨血小板源性生长因子(platelet-derived growth factor,PDGF)刺激成骨细胞,对磷酸化细胞外信号调节激酶1/2(phosphorylation extracellular signal-regulated kinase1/2,pERK1/2)位置的影响。方法出生3d清洁级健康小鼠10只,雌雄不拘,体重6～9g。取小鼠颅骨,分离培养原代成骨细胞。取第6代成骨细胞,1%血清培养液培养12h后,随机分成经10μmol/L PP2处理30min组(实验组)和未处理组(对照组),每组再随机分成2个亚组:其中一组用PDGF(20ng/ml)刺激10min,另一组不用PDGF刺激,采用免疫组织化学染色检测pERK1/2分布。另取第6代成骨细胞,当细胞生长至80%融合时,用细胞刮随机分成2组,一组用10μmol/L PP2预处理30min(实验组),另一组不用PP2作用(对照组),再用20ng/ml PDGF培养12h,采用划痕愈合法检测PP2对成骨细胞在PDGF刺激下迁移能力的影响。另取第6代成骨细胞,调整细胞浓度1×106/ml,随机分成2组,分别经DMSO(对照组)和10μmol/L PP2(实验组)预处理30min,每组再随机分成2个亚组:其中一组用PDGF(20ng/ml)刺激10min,另一组不用PDGF剌激,采用Western blot检测细胞骨架蛋白内pERK1/2活性。结果免疫荧光染色结果显示,PDGF促进pERK1/2定位于成骨细胞的局部黏附和细胞核内;而PP2显著抑制了由PDGF刺激引起的pERK1/2定位于成骨细胞的局部黏附,但并不影响pERK1/2定位于细胞核内。细胞划痕愈合实验显示,PP2明显抑制了由PDGF所诱导的成骨细胞迁移。Western blot检测结果显示,PP2明显抑制了由PDGF所诱导的成骨细胞局部黏附内ERK1/2的磷酸化。结论PDGF通过激活Src活性,促进pERK1/2定位于成骨细胞的局部黏附内;PP2通过抑制pERK1/2定位于成骨细胞的局部黏附,从而抑制由PDGF所诱导的成骨细胞迁移。
Objective To study the function of platelet-derived growth factor （PDGF） in phosphorylation extracellular signal-regulated kinase 1/2 （pERK1/2） localization in osteoblasts. Methods inducing 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 （20ng/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 μmol/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. Results 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.Conclusion Src activation is required for pERK1/2 translocalizatlon to focal adhesions and osteoblasts migration.
出处
《中国修复重建外科杂志》
CAS
CSCD
北大核心
2007年第11期1179-1183,共5页
Chinese Journal of Reparative and Reconstructive Surgery
基金
国家自然科学基金资助项目(30670876)~~
