补肾中药对人成骨细胞钙离子摄取和钙化能力的影响

Effects of kidney-tonifying Chinese herbal drugs on human osteoblast Ca^(2+) intake and minera lization in vitro

目的研究补肾中药对人成骨细胞增殖、分化、钙摄取和矿化能力的影响。方法用酶消化法从成人髂骨松质骨,分离人成骨细胞,以细胞形态学、碱性磷酸酶染色和体外成骨能力的检测鉴定成骨细胞。在成骨细胞培养液内分别加入含有高、中、低不同浓度的补肾中药鼠血清,以正常鼠血清作为对照。培养30min后,进行成骨细胞胞内钙离子变化的检测;培养3d后,用流式细胞仪检测成骨细胞增殖、分化情况;培养28d后,进行Von-Kossa染色,观察成骨细胞钙化结节的形成情况。结果与对照组相比,培养30min后,高、中、低3种浓度的补肾中药血清均能提高成骨细胞钙离子摄取能力;培养3d后,中药组处于增殖期的成骨细胞明显升高,和对照组相比具有明显的统计学差异。培养28d后,中药组成骨细胞钙化结节形成的数量也相对增多,其中,高浓度补肾中药血清处理组的钙化结节的数量与对照组相比有明显增加(P<0.05)。结论补肾中药血清能提高成骨细胞钙离子摄取能力,刺激成骨细胞增殖分化,增加成骨细胞钙化结节的形成。

Objective To study the effects of kidney-tonifying Chinese herbal drugs on Ca2+ intake and mineralization of human osteoblasts in vitro. Methods Human osteoblasts were isolated from the iliac trabecular bone followed by purification and culture at 37 ℃ with 5% CO2. The cells were identified by cell morphology, calcilum nodule formation and alkaline phosphatase (ALP) activity assay. The third passage of the cultured osteoblasts were treated with 10% scrum from rat fed with the decoction of the kidney-tonifying Chinese herbal drugs of different concentrations for 30 min, 3 d and 28 d, respectively. The cells treated with 10% rat serum without the drugs served as the control. Flow cytometry was used to observe the changes in cell proliferation and intracellular Ca2+ concentration, and von Kossa staining employed for quantification of the mineral nodules. Results The osteoblasts obtained were positive for ALP staining and could form calcium nodules in vitro. Flow cytometry showed that the drugs at different concentrations all increased Ca2+ influx, as compared with the control cells. The drugs also increased the relative proliferation index of the osteoblasts, and high concentration of the drugs resulted in greater number of the mineral nodules in the osteoblasts (P<0.05). Conclusion The kidney-tonifying Chinese derbal drugs may increase Ca2+ influx and stimulate proliferation and differentiation of adult osteoblasts in vitro.