卫生研究

目的 观察细颗粒物(fine particulate matter, PM_2.5)暴露对小鼠骨量、微观结构、生物力学性能以及小鼠骨髓间充质干细胞(bone marrow mesenchymal stem cells, BMSCs)成骨分化能力的影响。方法 选取16只8周龄C57BL/6J小鼠，随机分为对照组(NS组)和PM_2.5染毒组(PM组),每组8只，NS组给予生理盐水，PM组给予14 mg/kg PM_2.5悬浊液，每次50μL,每3天气管灌注染毒一次。10周后处死，取小鼠肺部进行HE染色，左侧胫骨进行Micro CT检测，右侧胫骨进行HE染色，免疫组织化学染色检测I型胶原(collagen I,Col I)、骨保护素(osteoprotegerin, OPG)和核因子-κB受体激活因子配体(receptor activator of nuclear factor-κB ligand, RANKL)蛋白表达和抗酒石酸酸性磷酸酶(tartrate resistant acid phosphatase, TRAP)染色。取双侧股骨提取原代BMSCs,成骨诱导后进行碱性磷酸酶(alkaline phosphatase, ALP)染色，茜素红染色，以及Real-time PCR检测骨钙素(osteocalcin, OCN)、ALP、OPG和RANKL mRNA的表达情况，并进行生物力学试验。结果 与NS组相比，PM组小鼠肺泡结构破坏，并有大量炎症细胞聚集，提示PM_2.5染毒操作成功。Micro CT结果显示PM组小鼠松质骨密度(bone mineral density, BMD)和骨体积分数(bone volume/total volume, BV/TV)分别为(276.959±15.152)mg/cm³和0.208%±0.009%,NS组为(316.709±28.205)mg/cm³和0.236%±0.019%,PM组均低于NS组(P<0.05),但骨小梁数量(trabecular number, Tb.N)、骨小梁厚度(trabecular thickness, Tb.Th)和骨小梁分离度(trabecular separation, Tb.Sp)等参数差异无统计学意义(P>0.05)。胫骨HE染色结果显示，NS组骨小梁厚且致密均匀。PM组骨小梁纤细，数量减少，间距变宽，排列稀疏。PM组ColⅠ(0.023±0.009)和OPG(0.036±0.010)与NS组(0.079±0.007,0.059±0.012)相比表达增多，RANKL(0.036±0.006)与NS组(0.022±0.002)相比表达减少(P<0.05);PM组TRAP阳性颗粒数量增多。小鼠BMSCs成骨诱导后实验结果显示，与NS组相比，PM组ALP阳性细胞数量减少，钙结节数量减少。PM组ALP、OCN和OPG mRNA(0.375±0.021,0.585±0.088,0.768±0.112)相对表达与NS组(1.001±0.043,1.006±0.132,1.002±0.086)相比明显减少，RANKL mRNA(1.278±0.118)相对表达相较于NS组(1.001±0.057)增多(P<0.05)。生物力学实验结果显示，NS组最大挠度0.337±0.031 mm, PM组最大挠度0.258±0.041 mm,与NS组相比，PM组最大挠度明显下降(P<0.05),最大应力和最大载荷有下降趋势，但差异无统计学意义(P>0.05)。结论 PM_2.5 染毒10周即可影响小鼠骨骼健康，其机制可能与破骨活性增强，BMSCs的成骨分化能力受到抑制有关。

OBJECTIVE To observe the effects of exposure to fine particulate matter(PM_2.5) on bone mass, microstructure, biomechanical properties, and osteogenic differentiation ability of bone marrow mesenchymal stem cells(BMSCs) in mice.METHODS A total of 16 C57BL/6J mice aged 8 weeks were randomly divided into control group(NS group) and PM_2.5 exposure group(PM group). NS group was given normal saline, PM group was given 14 mg/kg PM_2.5 suspension, 50 μL, poisoning every 3 day. After 10 weeks, the lungs of mice were taken for HE staining, and the left tibia was taken for Micro CT detection to analyze parameters related to cancellous and cortical bone. The right tibia was taken for HE staining to observe changes in bone trabeculae. Immunohistochemical staining was used to detect type I collagen(Col I), osteoprotegerin(OPG), and nuclear factor-κB receptor activating factor ligand(RANKL) protein expression, tartrate resistant acid phosphatase(TRAP) staining for detection of osteoclasts. Extract primary BMSCs from bilateral femurs, induce osteogenesis, and then perform alkaline phosphatase(ALP) staining to detect ALP activity, alizarin red staining to detect bone mineralization ability, real-time PCR to detect osteocalcin(OCN), ALP, OPG, and RANKL mRNA expression, and biomechanical testing to test the mechanical properties of the femur.RESULTS Compared with the NS group, the pulmonary alveolar structure of the PM group mice was disrupted and a large number of inflammatory cells gathered. Prompt for successful PM_2.5 poisoning operation. Micro CT result showed that the bone mineral density(BMD) and bone volume fraction(BV/TV) of the PM group mice were 276.959±15.152 mg/cm³ and 0.208%±0.009%, respectively. The NS group had 316.709±28.205 mg/cm³ and 0.236%±0.019%, respectively. The PM group was lower than the NS group(P<0.05), but the trabecular number(Tb.N) There was no statistically significant difference in parameters such as trabecular thickness(Tb.Th) and trabecular separation(Tb.SP)(P>0.05). The HE staining result of the tibia showed that the trabeculae in the NS group were thick, dense, and uniform. The bone trabeculae in the PM group were slender, with a decrease in number, widened spacing, and sparse arrangement. The expression of Col I(0.023±0.009) and OPG(0.036±0.010) in the PM group increased compared to the NS group(0.079±0.007, 0.059±0.012), while the expression of RANKL(0.036±0.006) decreased compared to the NS group(0.022±0.002)(P<0.05); The number of TRAP positive particles increased in the PM group. The experimental result after osteoinduction of BMSCs in mice showed that compared with the NS group, the PM group had a decrease in the number of ALP positive cells and a decrease in the number of calcium nodules. The relative expression of ALP, OCN, and OPG mRNA in the PM group(0.375±0.021, 0.585±0.088, 0.768±0.112) was significantly reduced compared to the NS group(1.001±0.043, 1.006±0.132, 1.002±0.086), while the relative expression of RANKL mRNA(1.278±0.118) was increased compared to the NS group(1.001±0.057)(P<0.05). The biomechanical experimental result showed that the maximum deflection of the NS group was 0.337±0.031 mm, while the maximum deflection of the PM group was 0.258±0.041 mm. Compared with the NS group, the maximum deflection of the PM group decreased significantly(P<0.05), and the maximum stress and maximum load showed a decreasing trend, but the difference was not statistically significant(P>0.05).CONCLUSION After 10 weeks of exposure to PM_2.5, it can affect the bone health of mice, and its mechanism may be related to increased osteoclast activity and inhibition of the osteogenic differentiation ability of BMSCs.