血清素对体外培养大鼠成骨细胞增殖分化和矿化功能的影响

Effects of serotonin on proliferation, differentiation and mineralization of cultured rat osteoblasts in vitro

目的探讨不同浓度血清素对体外培养大鼠成骨细胞（OBs）增殖、分化和矿化功能的影响。方法在新生SD人鼠颅骨OBs培养液中分别加入不同浓度血清素：0mol／L组（空白对照组）、10^-9mol／L组、10^-8mol／L组、10^-7mol／L组、10^-6mol／L组、10^-5mol／L组，观察不同浓度血清素对OBs的增精能力（CCK-8法）、分化能力（Western法检测碱性磷酸酶蛋白水平、pNPP法检测碱性磷酸酶活性和ELISA法榆测骨钙素蛋白表达水平）和矿化能力（茜素红染色）的影响。并用荧光定最聚合酶链反应方法检测OBs分化甲、晚期血清素受体亚型的mRNA表达水平．结果不同浓度血清素均可抑制OBs的增殖、分化和矿化，这种抑制作用在低浓度时具有时间依赖性，而在高浓度时作用减弱，呈现“V”形趋势。5-HT1A、5-HT1B、5-HT1D、5-HT2A、5-HT2B、5-HT2C等受体在OBs上均有表达，其中5-HT2A和5-HT1B受体是表达最多的两种受体亚型。结论体外OBs表达血清素受体，血清索呵抑制OBs的增殖、分化和矿化，提示血清素能够凋节骨代谢。

Objective To investigate the effects of serotonin at different concentrations on prolifration, differentiation and mineralization of cuhured primary rat osteoblasts （OBs） in vitro. Methods The cuhure medium with different serotonin concentrations （from 10 9 to l0s tool/l,） and the osteoblasts fl＇om the skull of newborn SD rats were mixed to observe respectively the proliferation （using CCK-8 method）, the differentiation （using detections of alkaline phosphatase osteocalcin protein levels and alkaline phosphatase activity）, and the mineralization （using ARS staining and OD562） of the OBs. The experiments were perfimned in 6 groups： blank control, 10-9mol/L, 10-8mol/L, 10.7 mol/L, 106 mol/L and 10-5 mol/L groups. In addition, real-time RT-PCR was used to detect the mRNA expression levels of serotonin receptor subtypes both in early and late differentiation stages of OBs. Results Different concentrations of serotonin inhib- ited the proliferation, differentiation and mineralization of OBs. The inhibitory effect of serotonin at low concen- trations was dose-dependent, but it was relieved at high concentrations, displaying a V-shaped trend. All of the six 5-ftT receptor subtypes （5-HT1A, 5-HTIB, 5-HTID, 5-HT2A, 5-HT2B, and 5-H＇I2C receptors） were found to exist in OBs, a,nong which 5-H32A and 5-HT1B receptor subtypes had the highest expressions. Conclusion 5-liT receptors are expressed in osteoblasts in vitro and serotonin can inhibit proliferation, difikerentiation and mineralization of ostenblasts, suggesting that serotonin can modulate bone metabolism.