骨质疏松小鼠模型骨密度及骨代谢等相关指标的变化

Changes of bone mass density and biochemical indexes of bone metabolism in mice with osteoporosis

目的研究骨质疏松小鼠模型骨密度(BMD)及骨代谢等指标的变化规律,为揭示骨质疏松症的机理提供新的实验方法及思路。方法 C57BL/6J雌性小鼠随机分为模型组(Ovx组,n=25)和假手术组(Sham组,n=5),建立骨质疏松模型。以微计算机断层扫描技术(Micro-CT)测量其BMD,根据测量结果选取模型组中BMD最低和最高的5只小鼠分别作为低骨密度组(L组)和高骨密度组(H组)。应用酶联免疫吸附法(ELISA)检测其血清雌二醇(E^2)水平和碱性磷酸酶(ALP)活性;提取骨质疏松小鼠的骨髓间充质干细胞(BMSCs)并鉴定其表面抗原;通过使用茜素红染色观察钙化结节形成情况,对比不同组小鼠BMSCs的骨矿化能力。结果 L组、H组和Sham组的BMD分别为(297.82±10.22)、(365.26±3.25)、(442.76±21.96)mg/cm^2,各组间比较差异有统计学意义(P<0.05);Ovx组、L组、H组的E^2水平分别为(142.75±25.26)、(142.49±17.01)、(133.54±24.58)pg/ml,均低于Sham组的(240.38±36.54)pg/ml,差异有统计学意义(P<0.05);L组、H组、Sham组的ALP活性分别为(221.82±33.58)、(155.65±14.99)、(117.30±20.96)U/L,各组间小鼠血清ALP活性差异有统计学意义(P<0.05);流式细胞仪分析显示小鼠BMSCs不表达的表面标记包括:内皮细胞表面标记CD31(阳性表达率:1.4%),巨噬细胞及单核细胞表面标记CD11b(阳性表达率:0.8%),白细胞及淋巴细胞表面标记CD45(阳性表达率:0.3%),而表达黏附分子和间充质干细胞表面标记Ly-6A/E(阳性表达率:85.0%)。茜素红染色实验反应强弱的顺序为Sham组>H组>L组>Blank组。结论受个体遗传因素的影响,骨质疏松小鼠可表现出具有明显差异的BMD和骨代谢水平,其机制可能与BMSCs成骨分化基因的差异表达有关。

Objective To investigate the changing characteristics of bone mineral density（BMD）and biochemical indexes of bone metabolism in mice with osteoporosis,and provide a new method and disease model to reveal the genetic mechanism of osteoporosis. Methods Osteoporotic mouse model was established by applying ovariectomized C57BL/6J female mice,which BMD were measured through micro computed tomography（Micro-CT）subsequently. Both 5 mice with lowest and highest BMD were selected and divided into two separate groups：Group L and Group H. Serum estradiol（E-2）levels and alkaline phosphatase（ALP）activity were examined by enzyme linked immunosorbent assay（ELISA）. Bone mesenchymal stem cells（BMSCs）were isolated from osteoporotic mice,whose expression of surface antigens were analyzed by flow cytometry. Mineralized nodules observed from Alizarin red staining were used to compare the ability of bone mineralization of BMSCs in the mice from different groups. Results The BMD of group L, H and Sham were（297.82±10.22）,（365.26±3.25）and（442.76±21.96）mg/cm^2. Differences between the groups were statistically significant（P〈0.05）. The level of E-2 of group Ovx, L and H were（142.75±25.26）,（142.49±17.01） and（133.54±24.58）pg/ml, which were significantly lower than group Sham：（240.38±36.54） pg/ml（P〈0.05）. The ALP activity of group L, H and Sham were（221.82±33.58）,（155.65±14.99） and（117.30±20.96） U/L. Differences between the three groups were significant（P〈0.05）. Flow cytometry analysis showed that the BMSCs are negative for CD31（endothelial cell marker,1.4%）, CD11b（macrophage and monocyte marker,0.8%） and positive for CD45（pan-leukocyte marker,0.3%）, but positive for Ly-6A/E（surface adhesion molecule and MSC marker, 85.0%）. The activity order of Alizarin red reaction is Sham H 〉L〉 Blank. Conclusion Due to their individual genetic background,biochemical indexes of bone metabolism and BMD in osteoporotic mice show a significant difference,and its mechanism might be associated with differential expression of bone differentiation related gene of BMSCs.