低剂量 X 线照射对破骨细胞分化及功能活性的作用机制

摘要

Objective:To explore the action mechanism of low-dose X-irradiation (LDI)on osteoclast differentiation and func-tional activity.Methods:RAW 264.7 cell lines were selected as osteoclast precursors,and they were induced to establish osteoclast models.The osteoclasts were randomly assigned into control group (0 cGy of LDI),irradiation group (10 cGy of LDI)and P2X7 -/- ir-radiation group (shRNA and 10 cGy of LDI).The concentration of adenosin triphosphate (ATP)in culture medium of each group was detected 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 (MMP-9 )gene mRNA were deter-mined using real-time reverse transcription-polymerase chain reaction (RT-PCR).Results:LDI significantly improved the release of ATP in cell matrix in irradiation group and P2X7 -/- irradiation group,and ATP secretion increased markedly in irradiation group.Mor-phological experimental results indicated that LDI could improve the capabilities of osteoclast differentiation and bone resorption to some extent.When compared with control group,the mRNA expressions of P2X7 receptor and cathepsin K in irradiation group elevated dra-matically,whereas those in P2X7 -/- irradiation group decreased obviously (P <0.05).No significant difference was shown between ir-radiation group and control group by comparison to MMP-9 mRNA expression (P >0.05 ),but the MMP-9 mRNA expression in P2X7 -/- irradiation group decreased evidently (P <0.05).Conclusion:LDI can improve the capabilities of osteoclast activity,differ-entiation and bone resorption by ATP coupling P2X7 receptor.%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 P2X7－／－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 P2X7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 P2X7－／－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 P2X7受体及 cathepsin K mRNA 表达明显升高，而 P2X7－／－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 P2X7－／－照射组 MMP-9 mRNA 表达明显降低（P ＜0．05）。结论：LDI 通过 ATP 偶联 P2X7受体，从而提高破骨细胞的活性、分化及骨吸收能力。