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目的 观察不同剂量人甲状旁腺素氨基端 1 34片段 (hPTH1 34)连续刺激对SaoS 2人成骨肉瘤细胞系功能、信号传导的影响及机制。方法 对于对数生长期的SaoS 2细胞 ,采用 5、50、50 0和 5 0 0 0 μg/L浓度hPTH1 34连续刺激 8d。于刺激前、刺激中及刺激后 ,采用“金氏”化学法和放射免疫法测定培养液中碱性磷酸酶 (ALP)和骨钙素 (BGP)浓度 ;采用竞争蛋白结合法测定环磷酸腺苷(cAMP)浓度 ;采用逆转录 (RT) PCR对c fos基因表达水平进行半定量分析。结果 50 0 μg/LhPTH1 34刺激显著增高ALP水平 (P <0 .0 5 ,比对照组 ) ,促进成骨细胞分化。 5 0 0 0 μg/L刺激组BGP水平逐渐下降 (P值均 <0 .0 5 ,比对照组和刺激前 )。 50、50 0 μg/LhPTH1 34连续刺激组cAMP明显增高 (P值均<0 .0 5)。 5μg/L和 5 0 0 0 μg/L组cAMP无显著改变。 50、50 0 μg/L组c fos基因表达水平高于其他组。结论 hPTH1 34对成骨样细胞的影响与剂量显著相关 ,50及 50 0 μg/LhPTH1 34连续刺激显著促进成骨样细胞分化 ,增加骨骼的合成代谢。ALP、cAMP的水平及c fos基因表达对不同浓度hPTH1 34刺激的反应明显同步 ,提示hPTH1 34主要通过蛋白激酶A信号途径影响成骨细胞功能及c fos基因的表达 ,后者进一步在转录水平调节成骨细胞?
Objective To observe the effect of different doses of human parathyroid hormone 1 34 fragment (hPTH1 34) on the function and signal transduction of human osteosarcoma cell line SaoS and its mechanism. Methods For SaoS 2 cells in logarithmic growth phase, hPTH1 34 at concentrations of 5, 50, 50 and 50 μg / L were stimulated continuously for 8 days. The concentration of alkaline phosphatase (ALP) and osteocalcin (BGP) in the culture fluid were determined by “Kim’s” chemical method and radioimmunoassay before stimulation, during stimulation and after stimulation. The levels of cAMP (CAMP). Semi-quantitative analysis of c fos gene expression was performed by reverse transcription (RT) PCR. Results The stimulation of 50 0 μg / L hPTH1 34 significantly increased the level of ALP (P <0.05, compared with the control group), and promoted the differentiation of osteoblasts. The level of BGP decreased gradually in the 50 000 μg / L stimulation group (P <0.05, compared with the control group and before stimulation). 50, 50 0 μg / L hPTH1 34 continuous stimulation group cAMP was significantly higher (P value <0.05). There was no significant change of cAMP in 5μg / L and 500μg / L groups. The levels of c fos gene in 50 and 50 μg / L groups were higher than those in other groups. Conclusion The effect of hPTH1 34 on osteoblast-like cells is significantly correlated with the dose. Continuous stimulation with 50 and 50 μg / L hPTH1 34 significantly promoted osteoblast-like cell differentiation and increased bone anabolism. ALP, cAMP levels and c fos gene expression of hPTH1 34 stimulation of different concentrations of the response was synchronized, suggesting that hPTH1 34 mainly through the protein kinase A signaling pathway affect osteoblast function and c fos gene expression, the latter is further regulated at the transcriptional level osteoblast?