甲状旁腺素氨基端1-34片段不同作用方式对人成骨样细胞SaoS-2的影响

Effects of Different Human Parathyroid Hormone1-34Administration on SaoS-2Cells

目的观察人甲状旁腺素氨基端1-34片段(hPTH1-34)不同作用方式对人成骨样细胞(SaoS-2细胞)功能的影响,探讨hPTH1-34促进骨合成代谢的分子生物学机制。方法以48h为一个周期,每个周期分别采用50ng/ml的hPTH1-34进行连续刺激(刺激48h/周期)或间歇刺激(分别刺激1、3、6、12、24h/周期)SaoS-2细胞。采用“金氏”化学法、放射免疫法和竞争蛋白结合法测定碱性磷酸酶(ALP)、骨钙素(BGP)和环磷酸腺苷(cAMP)浓度;c-fos基因表达水平采用逆转录-聚合酶链反应技术(RT-PCR)进行半定量分析。结果hPTH1-34每周期间歇刺激3、6h呈时间依赖性显著增加ALP产生(与对照比,P<0.01;与连续刺激比,P<0.05或P<0.01)和cAMP浓度(与对照和连续刺激比,均P<0.05),并迅速促进c-fos基因表达。未观察到BGP对hPTH1-34刺激的明显反应。结论hPTH1-34对骨合成代谢的促进作用取决于间歇刺激方式,以每48小时间歇刺激3、6h作用最显著。蛋白激酶A信号途径是hPTH1-34发挥作用的主要途径,c-fos基因可能是重要的细胞内第三信使。

Objective To observe the effects of different human parathyroid hormone1-34(hPTH1-34)admi -nistration on SaoS-2cells,and explore the mechanism of bone formation improvement.Methods Each cycle covered48h.SaoS-2cells were continuously or intermittently stimulated by50ng/ml hPTH1-34for1,3,6,12,and24h in each cycle.Total RNA was extracted by Trizol kit.Alkaline phosphatase(ALP),osteocalcin or bone Gla-containing protein(BGP)and cyclic adenosine monophosphate (cAMP)levels were measured by chemical method,radioimmunoassay and competitive protein binding method,respectively.c-fos gene expression was semi -quantified by reverse transcription polymerase chain reaction(RT-PCR).Results ALP level was time -dependently increased in1,3and6h stimulation,especially in3and6h(compared with control,P<0.01;P<0.05or P<0.01compared with continuous stimulation).The cAMP level was time -dependently increased in3and6h incubation(P<0.05compared with control and continous stimulation).Intermittent hPTH1-34stimulation had more effects on cAMP level than continous action(P<0.001).hPTH1-34intermittent stimulation of1,3,and6h enhanced c-fos gene expression time -dependent ly.Conclusions Intermittent hPTH1-34stimulation has a stronger effect on osteoblast than continous action,especially in3,6h in each cycle intermittent stimulation.The synchronous responses of c-fos,ALP and cAMP to hPTH1-34suggest that hPTH1 - 34affect Saos - 2cells through cAMP dependent protein kinase A (PKA)pathway and c - fos gene paly an important role.