精脒对衰老兔骨髓间充质干细胞成骨分化的影响及机制研究

张广维; 侯颖; 吴方丽; 毕丽霞; 李爽; 党瑞杰; 田萧羽; 朱彪

doi:10.3969/j.issn.2095-5227.2021.08.013

精脒对衰老兔骨髓间充质干细胞成骨分化的影响及机制研究

Effects of spermidine on osteogenic differentiation of bone-marrow mesenchymal stem cells in senescent rabbit and its related mechanisms

摘要

摘要:
背景研究发现衰老兔骨髓间充质干细胞(bone-marrow mesenchymal stem cells，BMSCs)的成骨分化能力减弱，而精脒(Spermidine)对衰老相关疾病具有保护作用。
目的探讨精脒对衰老兔骨髓间充质干细胞成骨分化的影响及其作用机制。
方法复苏BMSCs并将其分为3组：对照组(Vehicle，培养基中不加入药物)，D-半乳糖诱导衰老组(D-gal，培养基中加入40 g/L D-gal诱导BMSCs衰老)，Spermidine干预组(Spermidine，培养基中加入40 g/L D-gal + 3 μmol/L Spermidine处理BMSCs)。试剂盒检测细胞内烟酰胺腺嘌呤二核苷酸(NAD⁺)与还原型NAD⁺(NADH)比例，MTT法检测细胞增殖活性。成骨诱导培养后，Western blot检测Sirt1蛋白的表达，qPCR检测成骨相关基因的转录水平。
结果与Vehicle组相比，D-gal组细胞内NAD⁺/NADH比例降低，BMSCs增殖减弱(P＜0.05)；Spermidine可提高细胞内NAD⁺/NADH比例并促进BMSCs增殖(P＜0.05)。成骨诱导培养后，D-gal抑制Sirt1表达并下调成骨相关基因Runx2、Osx和ALP的转录表达(P＜0.05)；而Spermidine可上调Runx2、Osx和ALP的转录表达(P＜0.05)。当采用siRNA抑制Sirt1表达后，Spermidine上调成骨相关基因转录表达的作用明显减弱(P＜0.05)。
结论 Spermidine可通过激活Sirt1促进衰老兔BMSCs成骨分化。

Abstract:
Background Osteogenic differentiation of senescent rabbit bone-marrow mesenchymal stem cells (BMSCs) are weakened with aging, and spermidine can attenuate age-associated diseases.
Objective To investigate the effects of spermidine on osteogenic differentiation of senescent rabbit BMSCs.
Methods BMSCs were divided into three groups: Vehicle, D-gal (D-galactose, 40 g/L D-gal), and Spermidine (40 g/L D-gal+3 μmol/L spermidine). NAD⁺/NADH kit was used to evaluate intracellular NAD⁺/NADH, and MTT method was used to detect cell viability. After cultured in osteogenic medium, western blotting assay was used to investigate the protein expression level of Sirt1, qPCR was used to detect osteogenic genes transcription.
Results Compared with the Vehicle group, NAD⁺/NADH level in D-gal group decreased and BMSCs growth was inhibited (P＜0.05); while NAD⁺/NADH level in spermidine group increased and BMSCs growth was promoted (P＜0.05). After cultured in osteogenic medium, D-gal decreased Sirt1 expression and down-regulated Runx2, Osx and ALP transcription (all P＜0.05); while spermidine up-regulated Runx2, Osx and ALP transcription (P＜0.05). However, when inhibiting Sirt1 using small interfering RNA, the effects of spermidine on promoting osteogenic genes transcription were blunted (P＜0.05).
Conclusion Spermidine can promote osteogenic differentiation of senescent rabbit BMSCs via activating Sirt1.

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