吡格列酮对体外培养大鼠破骨细胞样细胞分化及活性的影响

目的 观察吡格列酮对大鼠破骨细胞样细胞(OLC)分化及活性的影响,探讨PPARγ2活化与破骨细胞之间的关系.方法 用NF-кB受体活化因子配体(RANKL)及巨噬细胞集落刺激因子(M-CSF)协同诱导大鼠骨髓单个核细胞(BMC)向OLC分化,同时加入1、5、10μmol/L盐酸吡格列酮干预.应用RT-PCR分析OLC分化过程中PPARγ2及NF-кB受体活化因子(RANK)mRNA的表达,结合细胞染色及抗酒石酸酸性磷酸酶(TRAP)活性观察吡格列酮对OLC分化的影响,通过骨吸收陷窝分析及整合素β3(CD61)平均荧光强度的检测比较吡格列酮对OLC活性的影响.结果 (1)对OLC分化的影响:培养7 d时10μmol/L吡格列酮组与其它组相比OLC数量及TRAP活性明显减少(P<0.01或P<0.05),表明吡格列酮部分抑制OLC分化且此作用与剂量相关;RT-PCR结果显示在诱导分化早期,不同浓度吡格列酮呈剂量依赖性上调PPARγ2的表达水平,但随OLC的成熟其表达渐减弱,而RANK表达在5及10 p,mol/L吡格列酮干预组培养的各时间点均明显下调,与对照及1μmol/L干预组相比有显著差异(P<0.05或P<0.01);(2)对OLC活性的影响:骨吸收陷窝数量及吸收面积在吡格列酮5、10μmol/L组明显减少,与另两组相比差异显著(P<0.05或P<0.01),培养早期这两组细胞CD61平均荧光强度均明显下调(P<0.05或P<0.01).结论 吡格列酮激活PPAR田γ2转录活性可部分抑制大鼠骨髓OLC的分化及活性.

Effects of pioglitazone on differentiation and function of cultured osteoclast-like cells of rats

Objective To study the effect of pioglitazone on the differentiation and function of rat osteoclast-like cells (OLC), and to probe the relationship between activated PPARγ2 and osteoclasts. Methods On day 1 of OLC formation from nonadherent bone marrow ceils (BMC) obtained from rats induced by M-CSF and receptor activator of NF-кB ligand (RANKL), 1, 5 and 10μmol/L pioglitazone hydrochloride was added. RT- PCR was performed to determine the mRNA expressions of PPARγ2 and receptor activator of NF-кB (RANK) on day 3, 5 and 7 during incubation, the number of tartrate-resistant acid phosphatase (TRAP)-positive cells,the number of bone resorption pits and the ratio of its area on dentin slice were counted, the activity of TRAP and the mean fluorescence intensity of integrin β3 (CD61) of OLC were also measured. Results (1) The effect on the differentiation of OLC: The addition of pioglitazone at the start of the culture period induced a dose-dependent decrease in TRAP-positive OLC and the activity of TRAP (P < 0.01 or P < 0.05) ; the mRNA expression of PPARγ2 was up-regulated by 5 and 10 μmol/L pioglitazone in the early stage of incubation and attenuated with the maturation of OLC on the contrary, however, the expression of RANK was down-regulated by 5 and 10 μmol/L piolitazone in every stage of incubation (P < 0.05 or P < 0.01), combined with decrease in TRAP-positive OLC from day 3 by 10 μmol/L pioglitazone. (2) The effect on the function of OLC: the number of bone resorption pits and the ratio of its area on dentin slice were decreased in groups of 5 and 10 μmol/L pioglitazone (P < 0.01 or P < 0.05), no obvious change was noted in the group with 1 μmol/L pioglitazone compared with the control group; the mean fluorescence intensity of CD61 were down-regulated in groups of 5 and 10 μmol/L pioglitazone (P < 0.05 or P <0.01). Conclusion Activation of PPARγ2 pathway by pioglitazone could partially inhibit differentiation and function of OLC derived from rat BMC.