目的观察帕米膦酸二钠对人骨髓间充质干细胞(Mesenchymal stem cells,MSC)生物学特性的影响,以探索双磷酸盐导致骨组织损伤的机制。方法人骨髓MSC培养体系中加入不同浓度的帕米膦酸二钠,培养72 h后,MTT法测定490 nm光密度值,观察细胞增殖情况;培养1周后,流式细胞技术检测细胞表面分子表达。体外诱导MSC成骨分化,体系中加入1μg/mL帕米膦酸二钠,1周后PCR法测定细胞Runx-2表达水平,2周后组织化学法测定细胞内碱性磷酸酶活性,以细胞总蛋白量为参照,观察MSC成骨分化的差异。结果 MTT结果显示,在0.1~10μg/mL浓度范围内,帕米膦酸二钠抑制人骨髓MSC增殖,作用呈浓度依赖性,最低作用浓度为1μg/mL,72 h OD490显著低于对照组(P<0.01)。流式细胞检测显示,帕米膦酸二钠处理后,细胞仍均一表达CD44和CD73,不表达CD31和CD45。PCR及细胞化学结果显示,帕米膦酸二钠无促进MSC成骨分化作用,也未提高成骨诱导体系促分化效果。结论帕米膦酸二钠可抑制MSC增殖,不促进其体外成骨能力,可能是长期使用双磷酸盐导致骨损伤的机制之一。

Objective To observe the effects of Parmidronate disodium on the biological properties of human bone marrow mesenchymal stem cells (MSCs) to shed a light on the mechanisms underlying the damaging activity of alendronate on bone tissue. Methods Parmidronate disodium at graded doses was added into the culture of human bone marrow MSCs and the culture was maintained for 72 hours, MTT test was used to evaluate the cellular proliferation status. Also, the surface marker profile was observed after culture for one week. MSCs were cultured in the presence of parmidronate disodium (0.5μg/mL) for 2 weeks and the cellular activity of alkaline phosphatase was detected. The total cellular protein content was used as the internal reference. Results The results from MTT assay showed that Parmidronate disodium within a range of concentrations (0.1-10 μg/mL) inhibited human MSC growth in a dose-dependent manner;The lowest effective dose was 1.0μg/mL, and the value of OD490 from MSCs cultured in the presence of Parmidronate disodium for 72 hours was significantly lower than that of the counterparts without the addition of this drug. Flow cytometric analysis showed that parmidronate disodium-treated MSCs expressed homogenously CD44 and CD73 and were negative for CD31 and CD45. Furthermore, the cellular activity of alkaline phosphatase was unchanged after exposure to parmidronate disodium, and it did not take any effect on the differentiation-inducing activity of the standard osteogenic agents. Conclusion Parmidronate disodium could inhibit the proliferation of human bone marrow MSCs, and have not significant effect on their differentiation along osteoblast lineage. The growth inhibitory activity of parmidronate disodium might be one of the mechanisms responsible for its destructive action on bone tissue.