【摘要】 [背景] 900 MHz射频辐射是现代无线通信设备中的常用频率，其潜在健康效应备受关注，尤其对骨代谢的影响尚未完全阐明。[目的]探究900 MHz射频辐射对小鼠骨组织及成骨细胞衰老的影响及其剂量效应关系。[方法]体内实验选用3月龄雌性C57BL/6小鼠，随机分为5组：假暴露组、低剂量射频组(50μW·cm^-2)、中剂量射频组(150μW·cm^-2)、高剂量射频组(450μW·cm^-2)和D-半乳糖阳性对照组，每组10只。小鼠连续暴露于射频辐射或假暴露28 d，每天4 h。照射结束后，通过Micro-CT扫描评估股骨骨密度及骨微结构参数，包括骨体积、骨组织体积、骨体积分数、骨小梁数量、骨小梁分离度和骨小梁厚度；苏木精-伊红（HE）染色观察骨组织形态学变化；免疫组化检测骨代谢标志物骨保护素（OPG）和核因子-κB受体激活剂配体（RANKL）的表达；酶联免疫吸附（ELISA）测定血清中OPG、抗酒石酸酸性磷酸酶（TRACP-5b）以及炎症因子纤溶酶原激活物抑制剂-1（PAI-1）、白细胞介素-6（IL-6）和CXC趋化因子配体15（CXCL15）的水平；逆转录聚合酶链式反应分析骨组织中Tp53、Cdkn1a和Cdkn2a的mRNA表达水平。体外实验采用MC3T3-E1成骨前体细胞，分为5组：假暴露组、低剂量射频组(50μW·cm^-2)、中剂量射频组(150μW·cm^-2)、高剂量射频组(450μW·cm^-2)和过氧化氢阳性对照组。细胞暴露于射频辐射或假暴露5 d，每天4 h。通过光学显微镜观察细胞形态变化；CCK-8法检测细胞增殖活性；衰老相关β-半乳糖苷酶（SA-β-gal）染色评估细胞衰老程度；Western blot检测P53和P21蛋白表达水平；RT-PCR分析Tp53和Cdkn1a的mRNA表达水平。[结果]体内实验显示，各剂量射频组的小鼠股骨骨密度，骨微结构参数骨体积分数、骨小梁数量和骨小梁厚度均显著降低（P<0.05），其中，骨小梁分离度仅在150μW·cm^-2射频组中增大（P<0.05），骨网络结构疏松；HE染色揭示骨小梁数量减少、排列稀疏及骨髓腔脂肪沉积增加；免疫组化表明OPG表达下调（P<0.05）,RANKL表达上调（P<0.05）,ELISA检测发现150μW·cm^-2射频组和450μW·cm^-2射频组小鼠血清OPG水平显著降低（P<0.05）,50μW·cm^-2RF组呈降低趋势但差异无统计学意义（P > 0.05）,TRACP-5b水平升高（P<0.05），炎症因子PAI-1、IL-6和CXCL15表达增加（P<0.05）;RT-PCR结果显示骨组织中Tp53、Cdkn1a和Cdkn2a的mRNA表达显著上调（P<0.05）。体外实验表明，射频辐射导致成骨细胞扁平化、表面积增大，增殖活性降低（P<0.05）,SA-β-gal阳性细胞率增加（P<0.05）,P53和P21蛋白及其基因Tp53和Cdkn1a的表达上调（P<0.05）。[结论] 900 MHz射频辐射通过抑制骨形成、促进骨吸收及诱导成骨细胞衰老，显著破坏小鼠骨代谢平衡，加速骨组织衰老进程。150μW·cm^-2射频辐射对骨衰老的生物效应最为显著，体现非线性“窗效应”，提示射频辐射的健康风险需进一步关注。更多还原

【Abstract】 [Background] 900 MHz radiofrequency radiation（RF） is a commonly used frequency in modern wireless communication devices, and its potential health effects have drawn much attention,especially its impact on bone metabolism, which has not been fully clarified.[Objective] To investigate the effects of 900 MHz RF on the bone tissue and osteoblast senescence of mice, as well as the dose-effect relationship.[Methods] In vivo, 3-month-old female C57BL/6 mice were divided into five groups（n=10）: sham exposure, low-dose RF(50 μW·cm^-2),medium-dose RF(150 μW·cm^-2), high-dose RF(450 μW·cm^-2), and D-galactose positive control（D-gal）. Treatments were administered for4 h per day for 28 d. Bone mineral density（BMD） and microstructure, including bone volume（BV）, tissue volume（TV）, bone volume fraction（BV/TV）, trabecular number（Tb.N）, trabecular separation（Tb.Sp）, and trabecular thickness（Tb.Th）, were assessed by Micro-CT; bone morphology was examined after hematoxylin and eosin（HE） staining; osteoprotegerin（OPG） and receptor activator of nuclear factor kappa-κΒ ligand（RANKL） expression was detected by immunohistochemistry; serum OPG, tartrate-resistant acid phosphatase 5b（TRACP-5b）, plasminogen activator inhibitor-1（PAI-1）, interleukin-6（IL-6）, and C-X-C motif chemokine ligand 15（CXCL15） levels were measured by enzyme-linked immunosorbent assay（ELISA）; mRNA expression of Tp53, Cdkn1a, and Cdkn2a in bone tissue was analyzed by reverse transcription polymerase chain reaction（RT-PCR）. In vitro, MC3T3-E1 pre-osteoblasts were grouped into sham, low-dose RF(50 μW·cm^-2), medium-dose RF(150 μW·cm^-2), high-dose RF(450 μW·cm^-2), and H2O2 control, groups, and were exposed for 4 h per day for 5 d. Cell morphology was observed by microscopy; viability was tested by cell counting kit-8（CCK-8）; senescence was evaluated by senescence-associated β-galactosidase（SA-β-gal） staining; P53 and P21 protein expression was detected by Western blot; Tp53 and Cdkn1a mRNA levels were measured by RT-PCR.[Results] In vivo, RF at each dose significantly reduced the BMD of the mice’s femurs and the bone microstructure parameters, such as BV/TV, Tb.N, and Tb.Th（P < 0.05）. Among them, Tb.Sp only increased in the 150 μW·cm^-2 RF group（P < 0.05）, with a looser bone network; fewer, sparser trabeculae and increased marrow fat were observed after HE staining; down-regulated OPG and up-regulated RANKL expression levels were observed by immunohistochemistry; the ELISA test revealed that the serum OPG levels in the 150 μW·cm^-2 RF group and the 450 μW·cm^-2 RF group of mice were significantly decreased（P < 0.05）, while the indicator in the 50 μW·cm^-2 RF group showed a decreasing trend but the difference was not statistically significant（P > 0.05）, TRACP-5b rose, and PAI-1, IL-6, and CXCL15 levels increased（P < 0.05）; the RT-PCR results showed that Tp53, Cdkn1a, and Cdkn2a mRNA expression was upregulated（P < 0.05）. In vitro, radiofrequency radiation induced cell flattening, reduced viability（P < 0.05）, increased SA-β-gal-positive cells（P < 0.05）, and upregulated P53, P21, Tp53, and Cdkn1a expression（P < 0.05）.[Conclusion] 900 MHz RF disrupts bone metabolism in mice by inhibiting bone formation, promoting resorption, and inducing osteoblast senescence, accelerating bone aging. The 150 μW·cm^-2 RF dose exhibits the most pronounced effect, reflecting a nonlinear “window effect,” highlighting potential health risks.更多还原