唑来膦酸对钛微粒诱导的破骨细胞前体细胞RAW 264.7分化的影响

摘要

[ABSTRACT]Objective:To observe the effect of zoledronic acid on the differentiation of titanium particle-induced osteoclast precursor cells RAW 264.7 to probe its possibility in the prevention and treatment of osteoporosis around prosthesis.Methods:RAW 264.7 cells were cultured in vitro.A preparation of titanium particles was made and the MTT method was used to detect the growth curve of RAW 264.7 cells in order to look for the best concentration of zoledronic acid for intervention.Cells Raw 264.7 was divided into three groups:the Ti particles group(0.1% volume ratio of titanium particles + 10% fetal bovine serum culture medium),the Ti+ zoledronic acid group(0.1% volume ratio of titanium particles + 10% fetal bovine serum culture medium + optimal concentration of zoledronic acid) and the control group(10% fetal bovine serum culture medium).The tartrate resistant acid phosphatase(TRAP) staining was used to observe the morphology of cells.The ELISA method was used to detect the concentration of RANK(receptoractivator of NF-κB).The biochemical method was used to determine the activity of TRAP in the supernatant of cells.The qPCR method was used for the detection of the expressions of TRAP,matrix metalloproteinase 9 (MMP-9),carbonic anhydraseⅡ(CAⅡ) and(NFATcl) mRNA;Western Blot was used to detect the expressions of TRAP,RANK,CtsK and proteins.Results:Titanium particles could stimulate RAW 264.7 to secrete osteoclast differentiation factors and promote the transformation of mononuclear cells to osteoclasts.Zoledronic acid could obviously make a down regulation for the expression of titanium particle-induced RAW 264.7 RANK and the expressions of TRAP,MMP-9,CAⅡ,NFATcl mRNA and proteins.The difference was statistically signiifcant (P< 0.05).Conclusion:Zoledronic acid can effectively inhibit the differentiation osteoclast precursor cells RAW 264.7,reduce the formation of osteoclast and make a down regulation of the phenotype of osteoclast and the transcription level of functional gene mRNA,which is expected to become a drug for the prevention and treatment of postoperative periprosthetic osteoporosis.%目的：观察唑来膦酸对钛微粒诱导的破骨细胞前体细胞RAW 264.7分化的影响，探讨其防治假体周围骨质疏松的可能性。方法：体外培养单核巨噬细胞白血病细胞RAW 264.7。制备钛微粒， MTT法检测绘制RAW 264.7细胞增殖曲线，寻找唑来膦酸最佳干预浓度。将RAW 264.7分为3组：Ti微粒组（0.1%体积比钛微粒+含质量分数为10%的胎牛血清培养基）、Ti+唑来膦酸组（0.1%体积比钛微粒+含质量分数为10%的胎牛血清培养基+最佳浓度唑来膦酸）和对照组（含质量分数为10%的胎牛血清的常规培养基）。采用抗酒石酸酸性磷酸酶（tartrate resistant acid phosphatase,TRAP）染色法观察细胞的形态。采用ELISA法检测培养液中细胞核因子κB活化因子受体（receptor activator of NF-κB,RANK）的浓度，生化法测定细胞上清液中TRAP活力，用qPCR法检测TRAP、基质外金属蛋白酶-9（matrix metalloproteinase-9,MMP-9）、碳酸酐酶Ⅱ（carbonic anhydraseⅡ,CAⅡ）、磷酸酶-活化T细胞核因子（NFATcl） mRNA的表达；Western Blot法检测TRAP、RANK、CtsK蛋白的表达。结果：钛微粒能刺激RAW 264.7分泌促破骨细胞分化的因子，并能促进单核细胞向破骨细胞转化。唑来膦酸可明显下调钛微粒诱导的RAW 264.7RANK的表达，以及TRAP、MMP-9、CAⅡ、NFATclmRNA和蛋白的表达，差异有统计学意义（P <0.05）。结论：唑来膦酸能有效抑制破骨细胞前体细胞RAW 264.7分化，减少破骨细胞的形成，下调RANK、TRAP等破骨细胞特异细胞表型和功能基因mRNA的转录水平，有望成为防治人工关节置换术后假体周围骨质疏松的药物。