低剂量X线照射对破骨细胞分化及功能活性的作用机制

Action mechanism of low-dose X-irradiation on osteoclast differentiation and functional activity

目的:探讨低剂量X线照射(LDI)对破骨细胞的分化及功能活性的作用机制。方法:选择RAW 264.7细胞株作为破骨前体细胞,诱导构建破骨细胞模型。将破骨细胞随机分为对照组(0 cGy LDI)、照射组(10 cGy LDI)和P2X_7^(-/-)照射组(shRNA,10 cGy LDI)。采用生物发光试剂盒检测各组培养基中三磷酸腺苷(ATP)的浓度,抗酒石酸酸性磷酸酶(TRAP)染色评估破骨分化成熟度,实时荧光定量逆转录多聚酶链反应(RT-PCR)法检测破骨细胞P2X_7受体、组织蛋白酶K(cathepsin K)及基质金属蛋白酶-9(MMP-9)基因mRNA的表达情况。结果:LDI显著提高了照射组和P2X_7^(-/-)照射组细胞基质中ATP的释放,照射组中ATP分泌更加显著。形态学实验提示,LDI在一定程度上提高了破骨细胞的分化和骨吸收能力。与对照组比较,照射组P2X_7受体及cathepsin K mRNA表达明显升高,而P2X_7^(-/-)照射组明显降低(P<0.05);照射组MMP-9 mRNA表达与对照组比较无显著差异(P>0.05),而P2X_7^(-/-)照射组MMP-9 mRNA表达明显降低(P<0.05)。结论:LDI通过ATP偶联P2X_7受体,从而提高破骨细胞的活性、分化及骨吸收能力。

Objective: To explore the action mechanism of low-dose X-irradiation ( LDI) on osteoclast differentiation and functionalactivity. Methods:RAW 264. 7 cell lines were selected as osteoclast precursors, and they were induced to establish osteoclastmodels. The osteoclasts were randomly assigned into control group (0 cGy of LDI) ,irradiation group ( 10 cGy of LDI) and P2X7 7 irradiationgroup ( shRNA and 10 cGy of L D I). The concentration of adenosin triphosphate ( ATP) in culture medium of each group wasdetected using bioluminescence kit. The maturity of osteoclast differentiation was evaluated by tartrate-resistant acid phosphatase(TRAP) staining,and expressions of P2X7 receptor, cathepsin K and matrix metalloproteinase-9 (M M P-9) gene mRNA were determinedusing real-time reverse transcription-polymerase chain reaction (RT-PCR). Results: LDI significantly improved the release ofATP in cell matrix in irradiation group and P2X? irradiation group, and ATP secretion increased markedly in irradiation group. Morphologicalexperimental results indicated that LDI could improve the capabilities of osteoclast differentiation and bone resorption to someextent. When compared with control group,the mRNA expressions of P2X7 receptor and cathepsin K in irradiation group elevated dramatically,whereas those in P2X? 7 irradiation group decreased obviously (P <0. 05) . No significant difference was shown between irradiationgroup and control group by comparison to MMP-9 mRNA expression ( P > 0. 05 ) , but the MMP-9 mRNA expression inP2X7 7 irradiation group decreased evidently (P<0.05). Conclusion:LDI can improve the capabilities of osteoclast activity, differentiationand bone resorption by ATP coupling P2X7 receptor.