RGD胰岛素对人破骨样细胞骨吸收功能与机制的研究

Effect of RGD-insulin on activities of bone resorption and the possible mechanism in human osteoclast-like cells in vitro

目的 以人骨巨细胞瘤中的破骨样细胞作为模型 ,探讨RGD(Arg Gly Asp)胰岛素抗骨吸收机制。 方法 培养细胞作骨吸收功能观察和抗酒石酸酸性磷酸酶 (TRAPase)染色鉴定。分别采用原位杂交、末端标记法和粘附实验观察RGD胰岛素和蛇毒蛋白对破骨样细胞中Ⅱ型碳酸酐酶(CAⅡ )基因表达、细胞凋亡和粘附能力的影响。 结果 破骨样细胞具有骨吸收功能 ,呈抗酒石酸磷酸酶 (TRAP)阳性。RGD胰岛素和蛇毒蛋白处理后 ,随药物浓度升高 ,破骨样细胞中CAⅡ表达下降 ,凋亡数目增多 ,药物浓度为 10 -5、10 -6和 10 -7mol/L时 ,蛇毒蛋白组细胞数分别为 (179± 3)、(2 38±11)、(35 1± 10 )个 /孔 ,RGD胰岛素分别为 (2 2 9± 2 0 )、(2 2 2± 2 1)、(2 6 5± 17)个 /孔 ,同浓度药物组间差异均有极显著性 (P <0 0 0 1) ,蛇毒蛋白组内差异有显著性 (P <0 0 5 ) ,而RGD胰岛素组内差异无显著性。RGD胰岛素抑制破骨样细胞粘附作用随药物浓度增加明显升高 ,药物浓度为 10 -5、10 -6、10 -7、10 -8mol/L时 ,其粘附破骨样细胞数目分别为 (16 5± 12 )、(16 0± 17)、(187± 2 4)、(2 71± 2 0 )个 /孔。与空白对照相比 ,除RGD胰岛素 10 -7mol/L组外 ,其余差异均有显著性 (P <0 0 5 )。 结论 RGD胰岛素同蛇毒蛋白一样 ,都能?

Objective In this study, we explored the mechanism of anti-bone resorption of RGD-insulin using osteoclast-like cells (OLCs) from giant cell tumor of bone as an in vitro model. Methods The function of bone resorption was observed and the staining for tartrate-resistant acid phosphatase (TRAPase) was identified. The carbonic anhydrase Ⅱ (CAⅡ) gene expression, apoptosis and the adhesion of OLC that treated by RGD-insulin and echistatin were determined by in situ hybridization, TUNEL staining and adhesion test, respectively. Results OLCs were TRAP positive and had bone resorptive function. After OLCs were treated with 10 -5, 10 -6, 10 -7mol/L RGD-insulin and echistatin respectively, the numbers of TRAP-positive multinucleated cells were (179±3), (238±11), (351±10) per well that treated with echistatin and (229±20), (222±21), (265±17) per well that treat with RGD-insulin.The expression of CAⅡ mRNA decreased, the number of OLCs apoptosis increased in a dose-dependent manner (P<0.001). After pretreated with 10 -5,10 -6,10 -7,10 -8mol/L RGD-insulin, the numbers of adhered OLCs were (165±12)?(160±17)?(187±24)?(271±20) per well. OLC adhesion was inhibited obviously with increasing concentrations of RGD-insulin and echistatin(P<0.05). Conclusions Both RGD-insulin and echistatin inhibited the expression of CAII mRNA and induced the OLC apoptosis in dose-dependent manner.