中国组织工程研究

中国组织工程研究 ›› 2020, Vol. 24 ›› Issue (19): 2985-2990.doi: 10.3969/j.issn.2095-4344.2042

• 骨髓干细胞 bone marrow stem cells • 上一篇    下一篇

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#br#

赵  桥1，2，杨  飞3，张成栋2，陈  硕2，孙  枭2，张  波3，肖东琴2，刘  康2，冯  刚1，2，段  可1   

  1. 1西南医科大学附属医院骨与关节科，四川省骨科置入器械研发与应用技术工程实验室，四川省泸州市  646000；2川北医学院第二临床医学院·南充市中心医院组织工程与干细胞研究所，四川省南充市   637000；3南充市中心医院骨科，四川省南充市  637000
  • 收稿日期:2019-05-08 修回日期:2019-05-22 接受日期:2019-08-07 出版日期:2020-07-08 发布日期:2020-04-07
  • 通讯作者: 冯刚，博士，教授，西南医科大学附属医院骨与关节科，四川省骨科置入器械研发与应用技术工程实验室，四川省泸州市 646000；川北医学院第二临床医学院·南充市中心医院组织工程与干细胞研究所，四川省南充市 637000 并列通讯作者：段可，博士，副教授，西南医科大学附属医院骨与关节科，四川省骨科置入器械研发与应用技术工程实验室，四川省泸州市 646000
  • 作者简介:赵桥，男，1993年生，西南医科大学在读硕士，主要从事脊柱外科方面的研究。
  • 基金资助:
    国家自然科学基金(81171472)；四川省科技厅应用基础项目(2016JY0123)；四川省科技厅应用基础项目(2018JY0100)；四川省卫计委科研项目(18PJ476)；四川省教育厅科研项目(17ZA0178)；南充市市校合作科研专项资金(NSMC20170310，NSMC20170453，18SXHZ0374，18SXHZ0377，18SXHZ0124)

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

Zhao Qiao1, 2, Yang Fei3, Zhang Chengdong2, Chen Shuo2, Sun Xiao2, Zhang Bo3, Xiao Dongqin2, Liu Kang2, Feng Gang1, 2, Duan Ke1   

  1. 1Department of Orthopedics and Arthrology, Affiliated Hospital of Southwest Medical University, Laboratory of Research and Development and Application of Orthopedic Devices; 2Second Clinical College of North Sichuan Medical College • Tissue Engineering and Stem Cell Research Institute of Nanchong Central Hospital; 3Department of Orthopedics, Nanchong Central Hospital
  • Received:2019-05-08 Revised:2019-05-22 Accepted:2019-08-07 Online:2020-07-08 Published:2020-04-07
  • Contact: Feng Gang, MD, Professor, Department of Orthopedics and Arthrology, Affiliated Hospital of Southwest Medical University, Laboratory of Research and Development and Application of Orthopedic Devices, Luzhou 646000, Sichuan Province, China; Second Clinical College of North Sichuan Medical College • Tissue Engineering and Stem Cell Research Institute of Nanchong Central Hospital, Nanchong 637000, Sichuan Province, China Duan Ke, MD, Associate professor, Department of Orthopedics and Arthrology, Affiliated Hospital of Southwest Medical University, Laboratory of Research and Development and Application of Orthopedic Devices, Luzhou 646000, Sichuan Province, China
  • About author:Zhao Qiao, Master candidate, Department of Orthopedics and Arthrology, Affiliated Hospital of Southwest Medical University, Laboratory of Research and Development and Application of Orthopedic Devices, Luzhou 646000, Sichuan Province, China; Second Clinical College of North Sichuan Medical College • Tissue Engineering and Stem Cell Research Institute of Nanchong Central Hospital, Nanchong 637000, Sichuan Province, China
  • Supported by:
    the National Natural Science Foundation of China, No. 81171472; the Applied Foundation Project of Science and Technology Department of Sichuan Province, No. 2016JY0123, 2018JY0100; the Scientific Research Project of Sichuan Health and Family Planning Commission, No. 18PJ476; the Scientific Research Project of Sichuan Provincial Department of Education, No. 17ZA0178; the Nanchong City-School Cooperative Research Special Funds, No. NSMC20170310, NSMC20170453, 18SXHZ0374, 18SXHZ0377, 18SXHZ0124

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摘要：

文题释义：

骨髓间充质干细胞：间充质干细胞是一种多能干细胞，最早于骨髓中发现，后来陆续在外周血、脂肪等组织中被分离出来。相比于其他来源的干细胞，骨髓间充质干细胞不仅具有自我更新及多向分化潜能，同时还能分泌某些有助于血管生成的细胞因子；在骨修复方面，其能在促成骨的同时促进局部毛细血管的再生，有较好的应用前景。

成骨分化：人体内干细胞在生长增殖过程中，受各种生物因素如细胞调控因子、激素水平以及细胞微环境中微量元素的影响，可向不同组织分化。影响成骨分化的因素包括降钙素、锌离子、铜离子、钙离子、骨形态发生蛋白2、生长分化因子5等，因此量化每一个影响因素对于骨组织工程具有重要意义。

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。

ORCID: 0000-0003-1353-2148(赵桥)

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

关键词: 骨髓间充质干细胞, 锌离子, 细胞增殖, 成骨分化, 矿化能力

Abstract:

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.

Key words: bone marrow mesenchymal stem cells, zinc ion, cell proliferation, osteogenic differentiation, mineralization

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