锌离子浓度可影响兔骨髓间充质干细胞的增殖与成骨分化

Zinc ion concentration affects proliferation and osteogenic differentiation of rabbit bone marrow mesenchymal stem cells

背景:锌离子是人体所必需的金属微量元素,在抑制破骨细胞活性和促进细胞成骨分化方面起着重要作用。然而,不同浓度锌离子对兔骨髓间充质干细胞增殖及成骨分化的影响却鲜有研究。目的:评价不同浓度锌离子对兔骨髓间充质干细胞增殖与成骨分化的影响,探索最适宜的锌离子浓度。方法:从乳兔长骨骨髓内提取骨髓间充质干细胞培养并传代,分为6组,即10-4,10-5,10-6,10-7,10-8 mol/L锌离子组以及空白对照组,采用CCK-8法检测细胞增殖活性并绘制生长曲线,碱性磷酸酶试剂盒评价细胞的成骨分化能力,活/死细胞染色表征细胞的生长活性,茜素红染色观察细胞的矿化能力,荧光定量PCR检测成骨相关基因的表达。结果与结论:①除10-4 mol/L锌离子组之外,随着时间延长,各组细胞数量呈增长趋势,且10-7 mol/L锌离子组在培养5 d后细胞数量最多,而10-4 mol/L锌离子组细胞数量最少;②细胞培养14 d后,当锌离子浓度为10-7 mol/L时,碱性磷酸酶活性最高,10-4 mol/L时碱性磷酸酶表达量最低;③活/死细胞染色显示10-7 mol/L锌离子组活细胞数量最多,10-4 mol/L锌离子组则几乎全为死细胞;④细胞培养14 d后,当锌离子浓度为10-7 mol/L时,可观察到明显的钙结节形成,且成骨相关基因表达水平最高;⑤结果表明,锌离子在一定浓度范围内,能有效促进兔骨髓间充质干细胞的增殖与成骨分化,且浓度为10-7 mol/L时,其促增殖、成骨诱导及促矿化能力最强。因此,在培养基中添加适宜浓度锌离子,有利于兔骨髓间充质干细胞的成骨分化。

BACKGROUND:Zinc ion is a necessary metal trace element in human body,which plays an important role in inhibiting osteoclast activity and promoting osteogenic differentiation.However,the effect of zinc ion at different concentrations on osteogenic differentiation of rabbit bone marrow mesenchymal stem cells has been rarely studied.OBJECTIVE:To evaluate the effects of zinc ion at different concentrations on the proliferation and osteogenic differentiation of rabbit bone marrow mesenchymal stem cells for exploring the appropriate zinc ion concentration.METHODS:Bone marrow mesenchymal stem cells were extracted from the long bone marrow of rabbits and passaged.The cells were divided into six groups,including 10-4,10-5,10-6,10-7 and 10-8 mol/L zinc ion groups,and blank control group.The cell proliferation activity was measured by Cell Counting Kit-8 and the growth curve was drawn.Alkaline Phosphatase Assay kit was used to evaluate the osteogenic differentiation ability of the cells,and the growth activity of the cells was shown by Live-Dead Cell Staining Kit.The mineralization ability of the cells was observed by alizarin red S staining.The expression of osteogenic genes was detected by fluorescence quantitative polymerase chain reaction.RESULTS AND CONCLUSION:The number of cells in each group showed an increasing trend with the prolongation of time except for the 10-4 mol/L zinc ion group.After 5 days of culture,the number of cells was highest in the 10-7 mol/L zinc ion group,but lowest in the 10-4 mol/L zinc ion group.Alkaline phosphatase activity was highest when zinc ion concentration was 10-7 mol/L,but lowest at the 10-4 mol/L zinc ion after cell culture for 14 days.The method of Live-Dead Cell Staining revealed the number of viable cells was highest in the 10-7 mol/L zinc ion group.All cells were almost dead in the 10-4 mol/L zinc ion group.Calcium nodule formation was visible,and the expression level of osteogenic genes was highest in the 10-7 mol/L zinc ion group after 14 days of cell culture.These findings suggest that zinc ion can effectively promote the proliferation and osteogenic differentiation of rabbit bone marrow mesenchymal stem cells in a certain concentration range.The promoting effects of zinc ion on proliferation,osteogenic induction and mineralization are strongest.Therefore,the addition of a suitable concentration of zinc ions in the medium is beneficial for the osteogenic differentiation of rabbit bone marrow mesenchymal stem cells.