金黄色葡萄球菌感染下骨细胞对破骨细胞分化形成的影响研究

Effect of Osteocytes on Osteoclast Differentiation Under the Infection of Staphylococcus Aureus

目的探讨金黄色葡萄球菌感染下,骨细胞对破骨细胞分化形成的影响。方法金黄色葡萄球菌感染骨细胞4 h后,灭活金黄色葡萄球菌,换液后细胞继续培养72 h。收集获得骨细胞源性的条件培养基(conditioned medium,CM),使用30%CM(诱导组)、等体积磷酸盐缓冲液(phosphate buffered saline,PBS;空白对照组)及核因子-κB受体活化因子配体(receptor activator of nuclear factor-κB ligand,RANKL;阳性对照组)对破骨细胞前体细胞(RAW264.7细胞)进行诱导培养,培养5 d后通过抗酒石酸酸性磷酸酶染色(tartrate resistant acid phosphatase,TRAP)计数破骨细胞分化形成的数目,并通过实时荧光定量聚合酶链式反应(real time-quantitative polymerase chain reaction,RT-qPCR)技术检测破骨细胞分化标志性基因的表达,评价其对破骨细胞分化形成的影响。结果诱导培养5 d后,TRAP染色显示:空白对照组(PBS组)基本无破骨细胞形成,条件培养基诱导组(30%CM组)及阳性对照组(RANKL组)均见破骨细胞形成,破骨细胞形成的数目经统计分析,各组间比较差异均具有统计学意义(P<0.05),RANKL组形成的破骨细胞数目多于30%CM组及PBS组。RT-qPCR结果显示,诱导培养5 d后,RANKL组及30%CM组破骨细胞分化形成的标志性基因:TRAP、calcitonin receptor(Cal R)、cathepsin K(Cathe K)、MMP-9的表达均高于PBS组,各组间比较差异均具有统计学意义(P<0.05)。结论金黄色葡萄球菌感染下,骨细胞可通过分泌某些活性因子促进破骨细胞分化形成增多,进而参与炎性骨破坏的病理过程。

Objective To investigate the effect of osteocytes on the differentiation and formation of osteoclasts under the infection of Staphylococcus aureus.Methods The osteocytes were infected with Staphylococcus aureus for 4 hours.Staphylococcus aureus was inactivated.After changing the culture medium,the cells were cultured for another 72 hours,and then,the culture medium was collected and named as conditioned medium(CM)derived from osteocytes.After that,the osteoclast precursor cells(RAW264.7 cells)were induced with this conditioned medium(Induction group),equal volume phosphate buffered saline(PBS,blank control group),and receptor activator of nuclear factor-κB ligand(RANKL,positive control group)for 5 days.In order to evaluate the effect of osteocytes on osteoclast differentiation,the number of osteoclasts was counted by tartrate resistant acid phosphatase(TRAP)staining,and the expression of osteoclast differentiation marker genes was detected by real time-quantitative polymerase chain reaction(RT-qPCR).Results After 5 days of induction,TRAP staining showed that there was less osteoclast formation in the blank control group(PBS group),but many osteoclasts were observed in the conditioned medium induction group(30%CM group)and the positive control group(RANKL group).After statistical analysis,the difference between the groups was statistically significant(P<0.05),and the number of osteoclasts formed in RANKL group was more than that in 30%CM group,more than that in PBS group.Moreover,RT-qPCR results showed that after 5 days induction,the expression of the marker genes of osteoclast differentiation in RANKL group and 30%CM group.Trap,calcitonin receptor,cathepsin K,MMP-9 were higher than that in PBS group.The difference between groups was statistically significant(P<0.05).Conclusion Under the infection of Staphylococcus aureus,osteocytes can promote the differentiation and formation of osteoclasts by secreting some active molecules,and then participate in the pathological process of inflammatory bone destruction.