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目的 :探讨肾上腺髓质素 (adrenomedullin ;ADM )对培养成骨细胞增殖的影响及其细胞内信号转导机制。方法 :分离培养胎大鼠颅骨成骨细胞 ,经ADM孵育 ,3 H 胸腺嘧啶 (3 H thymidine ,3 H TdR)参入法测定DNA合成 ,放射免疫法测定细胞 环磷酸腺甙 (cyclicadenosinemonophosphate,cAMP)含量 ,用底物蛋白磷酸法测定蛋白激酶A(proteinkinaseA ,PKA)、蛋白激酶C(proteinkinaseC ,PKC)、丝裂素活化蛋白激酶 (mitogen activatedproteinkinase ,MAPK )活性。结果 :随ADM浓度 (10 -12 ～ 10 -8mol·L-1)增加 ,成骨细胞3 H TdR参入呈线性增加(r =0 .998,P <0 .0 1)。ADM使成骨细胞cAMP含量和PKA、PKC、MAPK活性增加 (P <0 .0 5或P <0 .0 1)。PKC和MAPK激酶抑制剂能阻断ADM刺激成骨细胞3 H TdR参入增加的作用 ,而PKA和P38MAPK抑制剂对此无影响。结论 :ADM能够刺激成骨细胞增殖 ,激活PKC和MAPK/ERK(细胞外信号调节激酶extracellularsignal regu latedkinase) ,是其主要的细胞内信号转导途径 ,PKA、P38MAPK途径可能与此无关
Objective: To investigate the effect of adrenomedullin (ADM) on proliferation of cultured osteoblasts and its mechanism of intracellular signal transduction. Methods: The cultured rat calvarial osteoblasts were isolated and cultured. The DNA synthesis was determined by 3 H thymidine incorporation assay and the content of cyclic adenosine monophosphate (cAMP) was determined by radioimmunoassay The protein kinase A (PKA), protein kinase C (PKC) and mitogen activated protein kinase (MAPK) activity were measured by substrate protein phosphorylation assay. Results: With the increase of ADM concentration (10 -12 ~ 10 -8 mol·L-1), the 3 H TdR of osteoblasts increased linearly (r = 0.998, P <0.01). ADM increased the cAMP content and PKA, PKC and MAPK activities of osteoblasts (P <0.05 or P <0.01). PKC and MAPK kinase inhibitors blocked the increase of 3 H TdR incorporation in osteoblasts stimulated by ADM, whereas PKA and P38 MAPK inhibitors had no effect on this. CONCLUSION: ADM can stimulate the proliferation of osteoblasts and activate PKC and MAPK / ERK (extracellular signal regulatory kinase extracellular regu latedkinase), which are the main intracellular signal transduction pathways, and PKA and P38MAPK pathways may not be involved