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目的观察鲑鱼降钙素(calcitonin,CT)对离体培养数成骨细胞(osteoblast,OB)的作用,初步探讨CT对OB增殖以及胰岛素样生长因子I(Insulin-like Growth Factor-I,IGF-I)表达的影响,为促进骨修复和再生的临床治疗提供一定的理论依据。方法取新生24hSD乳鼠颅盖骨,采用酶消化法培养OB,经酶解消化后得到原代OB,进行体外培养并鉴定。将体外培养的OB用不同浓度(1×10-12～1×10-8mol/L)的CT处理,采用MTT法(噻唑蓝染色法)观察OB的增殖;Gomori钙-钻法检测碱性磷酸酶(ALP)的表达;RT-PCR方法观察胰岛素样生长因子-I(IGF-I)mRNA的表达。结果经细胞鉴定后证实其系OB;MTT法检测表明,CT浓度为1×10-12mol/L时就可以刺激OB增殖,且随CT浓度升高其增殖能力增加,呈剂量依赖关系;Gomori钙-钻法检测结果表明随着CT浓度的升高OB分化能力增强;经RT-PCR法检测表明,IGF-I mRNA可在乳鼠OB内稳定表达,半定量分析表明,随着CT浓度的升高,IGF-ImRNA的表达量逐渐增加。结论适当浓度的CT可以促进体外培养OB的增殖;部分机制可能与CT促进IGF-ImRNA表达的增加有关。
Objective To observe the effect of calcitonin (CT) on cultured osteoblasts (OB) in vitro and to investigate the effect of CT on the proliferation of OB and the expression of insulin-like growth factor-I (IGF- I) expression, to promote bone repair and regeneration of clinical treatment to provide a theoretical basis. Methods Calvaria of neonatal 24h SD rats were harvested, OBs were cultured by enzymatic digestion method, primary OBs were obtained after enzymolysis and digestion, and then cultured and identified in vitro. OB cultured in vitro with different concentrations (1 × 10-12 ~ 1 × 10-8mol / L) of CT treatment, MTT assay (thiazolyl blue staining) Observe the proliferation of OB; Gomori calcium - detection of alkaline phosphatase (ALP). The expression of insulin-like growth factor-I (IGF-I) mRNA was observed by RT-PCR. The results of cell identification confirmed that the Department of OB; MTT assay showed that CT concentration of 1 × 10-12mol / L can stimulate OB proliferation, and with the increase of CT concentration increased its proliferative capacity in a dose-dependent manner; Gomori calcium - The results of the drilling test showed that the OB differentiation ability was enhanced with the increase of CT concentration. The mRNA of IGF-I was stably expressed in OB rats by RT-PCR, and semi-quantitative analysis showed that with the increase of CT concentration High, the expression of IGF-I mRNA gradually increased. Conclusion CT at a proper concentration can promote the proliferation of OB cultured in vitro. Some mechanisms may be related to the increase of IGF-1 mRNA expression by CT.