锶离子对钛颗粒诱导破骨细胞活化的作用

Inhibitory effects of strontium on osteoclatogenesis induced by titanium particles

目的 观察锶离子（Sr2＋）对钛颗粒（Ti）诱导破骨细胞（OC）活化的影响。方法实验分为5组,即空白组、Ti组、核因子受体活化因子配体（RANKL）组、R＋Ti组、Sr2＋组。采用细胞增殖-毒性检测试剂盒（CCK-8）检测0.1 g/L Ti和不同浓度Sr2＋（0.5、1.0、2.0、5.0、10.0 mol/L）处理小鼠单核/巨噬细胞株RAW264.7细胞24、48、72 h后增殖活性,以0 1 g/L Ti和/或70μg/L RANKL和/或不同浓度Sr2＋诱导RAW264.7细胞,培养5 d后,用抗酒石酸酸性磷酸酶（TRAP）染色检测成熟OC数量,以逆转录-聚合酶链反应（RT-PCR）检测组织蛋白酶K（Cath-K）、TRAP、基质金属蛋白酶-9（MMP-9）和降钙素受体（CTR）mRNA含量,Westem blot法检测κB抑制蛋白α（IκBα）表达水平。结果 CCK-8结果,0.1 g/L Ti和不同浓度Sr2＋（0.5、1.0、2.0、5 0、10.0 mmol/L）对RAW264.7细胞增殖能力无影响。TRAP染色,R、R＋Ti组均可见大量的紫红色多核细胞,Ti组和Sr2＋组多核细胞较少,Sr2＋≥5 mmol/L时,TRAP阳性细胞数量[（323.33±43.84）、（146.67±33 99）个/孔],与R组[（453.33±33.99）个/孔]比较,差异有统计学意义（P〈0 05）。RT-PCR结果显示,Sr2＋组Cath-K、TRAP、MMP-9和CTR mRNA的相对表达量分别为2.76±0.66、2.69±0.57、2.10±0.34和1.72±0.32,与R＋Ti组比较（7.97±0.77、10.10±1.18、7.37±1.02和5.55±0.53）,差异有统计学意义（P〈0.05）。Western blot结果显示Ti能抑制IκBα的表达,Sr2＋加入后Ti的抑制效应不明显。结论 Sr2＋能有效地抑制Ti诱导的OC活化。

Objective To observe the effect of strontium （Sr2＋ ） on titanium （Ti） particles-in- duced osteoclastgogenesis from a murine macrophage cell line （RAW264. 7） in the presence of receptor ac- tivator of nuclear faetor-KB （NF-KB） ligand （RANKL）. Methods The experiment involved 5 groups ： black group, Ti group, R group, R ＋ Ti group and Sr2 ＋ group. The effect of Ti particles and （or） Sr2＋ on RAW264.7 cell viability was measured by using the cell counting kit-8. Tartrate resistant acid phosphatase （TRAP） staining was used to analyze the osteoclast formation. Quantitative real-time reverse transcriptionpolymerase chain reaction （RT-PCR） was used to detect the mRNA levels of cathepsin K （Cath-K） calcitonin receptor （CTR） , matrix metalloproteinase-9 （MMP-9） and TRAP. The inhibitory KBc＋ （IKBct） in NF-KB signaling pathway was determined by Western blotting. Results Sr2 ＋ （0. 5, 1.0, 2.0, 5.0, and 10.0 mmol/L） did not affect the viability of RAW264. 7 cells cultured with Ti particles. The mature osteo- clasts were obtained from RAW264. 7 cells cultured with RANKL and Ti particles for 5 days and detected by TRAP staining. The number of TRAP positive cells in 5 mmol/L Sr2＋ was （323.33 ±43.84）/well, which was significantly decreased as compared with R group [ （453. 33 ±33.99）/well] （P〈 0. 05）. RT-PCR demonstrated that mRNA levels of Cath-K, CTR, MMP-9 and TRAP were significantly greater in medium with RANKL and Ti particles than without RANKL and Ti particles. When 5 mmol/L Sr2 ＋ was added to the culture system for 5 days, the mRNA levels of Cath-K, TRAP, MMP-9 and CTR respectively were 2. 76±0. 66, 2. 69 ±0. 57, 2. 10±0. 34 and 1.72±0. 32, in combination with RANKL and Ti particles （7. 97 ± 0. 77, 10. 10 ± 1.18, 7.37 ± 1.02 and 5.55 ± 0. 53 ） （ P 〈 0. 05 ）. The results of Western blotting showed that the levels of IKBct were reduced at 15 min but increased again at 180 min after Ti particle treatment. Pretreatment with Sr2 ＋ significantly increased the expression of IKBct in response to Ti particle. Conclusion This study provides evidence that Sr2＋ can markedly inhibit Ti particle-induced oste-oclastogenesis by the down-regulation of NF-KB signaling pathway, and is a promisingly therapeutic candidate for the wear particle-induced osteolysis.