中华骨科杂志
中華骨科雜誌
중화골과잡지
CHINESE JOURNAL OF ORTHOPAEDICS
2011年
9期
970-975
,共6页
廖清船%徐康康%许静%张永%仇锦春%李天媛%王珊珊
廖清船%徐康康%許靜%張永%仇錦春%李天媛%王珊珊
료청선%서강강%허정%장영%구금춘%리천원%왕산산
细胞外信号调节MAP激酶类%成骨细胞%细胞分化%磷蛋白磷酸酶
細胞外信號調節MAP激酶類%成骨細胞%細胞分化%燐蛋白燐痠酶
세포외신호조절MAP격매류%성골세포%세포분화%린단백린산매
Extracellular signal-regulated MAP kinases%Osteoblasts%Cell differentiation%Phosphoprotein phosphatase
目的探讨骨髓间质干细胞(bone marrow mesenchymal stem cells,BMSCs)向成骨细胞分化过程中p38MAPK与ERK1/2的协同效应及其机制。方法 以成骨细胞分化添加剂诱导小鼠BMSCs向成骨细胞分化，测定碱性磷酸酶活性和钙沉积量。检测磷酸化p38MAPK和磷酸化ERK1/2(p-ERK1/2)的表达水平评估通路的激活状况。以SB203580或PD98059阻断p38MAPK或ERK1/2通路，观察对成骨细胞分化的影响。以SB203580或亚砷酸钠阻断或激活p38MAPK通路，观察p-ERK1/2的变化。以冈田酸抑制蛋白磷酸酯酶2A(protein phosphatases type 2A，PP2A)活性，观察p-ERK1/2的变化及对成骨细胞分化的影响。通过免疫共沉淀实验观察PP2A和ERK1/2间的结合及SB203580对结合的影响。结果成骨细胞分化添加剂诱导BMSCs向成骨细胞分化的过程伴有ERK1/2和p38MAPK通路的激活，SB203580剂量依赖性抑制成骨细胞分化，PD98059剂量依赖性增强成骨细胞分化。SB203580使p-ERK1/2表达增加，亚砷酸钠减弱其表达。冈田酸使p-ERK1/2表达增加，并使成骨细胞分化受到抑制。PP2A可直接与ERK1/2结合，SB203580使PP2A与ERK1/2的结合减弱。结论 p38MAPK可通过PP2A与ERKi/2产生协同效应，并调节BMSCs向成骨细胞分化。
目的探討骨髓間質榦細胞(bone marrow mesenchymal stem cells,BMSCs)嚮成骨細胞分化過程中p38MAPK與ERK1/2的協同效應及其機製。方法 以成骨細胞分化添加劑誘導小鼠BMSCs嚮成骨細胞分化，測定堿性燐痠酶活性和鈣沉積量。檢測燐痠化p38MAPK和燐痠化ERK1/2(p-ERK1/2)的錶達水平評估通路的激活狀況。以SB203580或PD98059阻斷p38MAPK或ERK1/2通路，觀察對成骨細胞分化的影響。以SB203580或亞砷痠鈉阻斷或激活p38MAPK通路，觀察p-ERK1/2的變化。以岡田痠抑製蛋白燐痠酯酶2A(protein phosphatases type 2A，PP2A)活性，觀察p-ERK1/2的變化及對成骨細胞分化的影響。通過免疫共沉澱實驗觀察PP2A和ERK1/2間的結閤及SB203580對結閤的影響。結果成骨細胞分化添加劑誘導BMSCs嚮成骨細胞分化的過程伴有ERK1/2和p38MAPK通路的激活，SB203580劑量依賴性抑製成骨細胞分化，PD98059劑量依賴性增彊成骨細胞分化。SB203580使p-ERK1/2錶達增加，亞砷痠鈉減弱其錶達。岡田痠使p-ERK1/2錶達增加，併使成骨細胞分化受到抑製。PP2A可直接與ERK1/2結閤，SB203580使PP2A與ERK1/2的結閤減弱。結論 p38MAPK可通過PP2A與ERKi/2產生協同效應，併調節BMSCs嚮成骨細胞分化。
목적탐토골수간질간세포(bone marrow mesenchymal stem cells,BMSCs)향성골세포분화과정중p38MAPK여ERK1/2적협동효응급기궤제。방법 이성골세포분화첨가제유도소서BMSCs향성골세포분화，측정감성린산매활성화개침적량。검측린산화p38MAPK화린산화ERK1/2(p-ERK1/2)적표체수평평고통로적격활상황。이SB203580혹PD98059조단p38MAPK혹ERK1/2통로，관찰대성골세포분화적영향。이SB203580혹아신산납조단혹격활p38MAPK통로，관찰p-ERK1/2적변화。이강전산억제단백린산지매2A(protein phosphatases type 2A，PP2A)활성，관찰p-ERK1/2적변화급대성골세포분화적영향。통과면역공침정실험관찰PP2A화ERK1/2간적결합급SB203580대결합적영향。결과성골세포분화첨가제유도BMSCs향성골세포분화적과정반유ERK1/2화p38MAPK통로적격활，SB203580제량의뢰성억제성골세포분화，PD98059제량의뢰성증강성골세포분화。SB203580사p-ERK1/2표체증가，아신산납감약기표체。강전산사p-ERK1/2표체증가，병사성골세포분화수도억제。PP2A가직접여ERK1/2결합，SB203580사PP2A여ERK1/2적결합감약。결론 p38MAPK가통과PP2A여ERKi/2산생협동효응，병조절BMSCs향성골세포분화。
Objective To study the synergistic effect of p38MAPK and ERK1/2 in bone marrow mesenchymal stem cells (BMSCs), and to explore their influence on osteogenic differentiation in BMSCs cultures. Methods Mouse BMSCs were cultured in phenol red-free α-MEM containing osteogenic supplements (OS) for inducing osteogenic differentiation. The temporal sequence of osteogenic differentiation in BMSCs cultures was assayed by measuring alkaline phosphatase activity (ALP) and calcium deposition gene expression. The activation of p38MAPK and ERK1/2 was detected by western blotting using phospho-specific MAP kinase antibody. BMSCs were treated with the inhibitor of p38MAPK pathway (SB203580) or ERK1/2 pathway (PD98059), and osteogenic differentiation was measured. BMSCs were treated with SB203580 or sodium arsenite (ARS), a strong activator of p38MAPK, and the phosphorylation of ERK 1/2 was measured. BMSCs were treated with PP2A inhibitor, Okadaic acid (OA), the phosphorylation of ERK1/2 and osteogenic differentiation were measured. lmmunoprecipitation was used to test the binding interaction between PP2A and ERK1/2, and the effect of SB203580 on the interaction. Results Treatment of BMSCs with osteogenic supplements resulted in activation of p38MAPK and ERK1/2 that coincided with osteogenic differentiation. Inhibition of p38MAPK activation by SB203580, blocked the osteogenic differentiation, whereas inhibition of ERK1/2 activation by PD98059, enhanced the osteogenic differentiation in a dose-dependent manner.SB203580 treatment resulted in increased ERK1/2 phosphorylation. By contrast, ARS treatment resulted in decreased ERK1/2 phosphorylation. Inhibition of PP2A by OA resulted in increased ERK1/2 phosphorylation. OS-induced osteogenic differentiation was also attenuated by PP2A inhibition. Immunoprecipitation confirmed the association of PP2A with ERK1/2 in BMSCs cultures, which was decreased by SB203580 treatment. Conclusion The present study demonstrates a synergistic effect between p38MAPK and ERK1/2 signaling pathways via PP2A in BMSCs cultures, which may regulate the osteogenic differentiation of BMSCs.
