《口腔颌面外科杂志》 ›› 2021, Vol. 31 ›› Issue (3): 150-155. doi: 10.3969/j.issn.1005-4979.2021.03.004

• 基础研究 • 上一篇    下一篇

过表达Sirt6促进脂多糖诱导大鼠骨髓间充质干细胞骨生成

王开(), 荆得宝, 于素平(), 刘雪阳   

  1. 海军军医大学附属公利医院口腔科,上海 200135
  • 收稿日期:2020-06-01 修回日期:2021-01-19 出版日期:2021-06-28 发布日期:2021-07-08
  • 通讯作者: 于素平,教授. E-mail: yusuping4939@sohu.com
  • 作者简介:

    王 开(1983—),男,河北石家庄人,主治医师,硕士. E-mail:

  • 基金资助:
    上海市浦东新区卫生健康委员会优秀青年医学人才培养项目(PWRq2017-18)

Overexpression of Sirt6 promotes bone formation of lipopolysaccharide induced rat bone marrow mesenchymal stem cells

WANG Kai(), JING Debao, YU Suping(), LIU Xueyang   

  1. Department of Stomatology, Gongli Hospital, Navy Medical University, Shanghai 200135, China
  • Received:2020-06-01 Revised:2021-01-19 Online:2021-06-28 Published:2021-07-08

摘要:

目的:探讨沉默信息调控因子6(silent information regulator 6,Sirt6)在炎症微环境下对骨髓间充质干细胞(bone marrow mesenchymal stem cells, BMSCs)的骨生成作用。方法:利用原代细胞贴壁法培养BMSCs,脂多糖(lipopolysaccharide,LPS)刺激BMSCs产生炎症反应;利用酶联免疫吸附试验(enzyme-linked immunosorbent assay,ELISA)检测促炎细胞因子:肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白细胞介素-1β(interleukin-1β,IL-1β)、IL-6的分泌。实验分为LPS组、LPS+空载体组及LPS+过表达Sirt6组,实时定量聚合酶链反应(real-time quantitative polymerase chain reaction,RT-qPCR)检测炎性状态下各组中Sirt6的mRNA表达;Western印迹法(Western blot)检测炎性状态下各组中Sirt6蛋白的表达水平;茜素红染色法检测炎性状态下各组BMSCs骨向分化中生成的钙结节。结果:培养出的BMSCs在LPS炎性刺激下可以产生炎性反应;炎症因子TNF-α、IL-1β、IL-6的含量均明显升高(P<0.05);成功构建了过表达Sirt6质粒载体并用其转染BMSCs;RT-qPCR和Western blot检测结果显示,LPS+过表达Sirt6组的Sirt6 mRNA和蛋白水平均明显高于LPS组和LPS+空载体组,并且LPS+过表达Sirt6组中成骨相关标志物骨钙素(osteocalcin,OCN)、碱性磷酸酶(alkaline phosphatase,ALP)、骨涎蛋白(bone sialoprotein,BSP)呈现高表达;茜素红染色检测结果显示,LPS+过表达Sirt6组中钙结节量也呈现高表达趋势。结论:过表达Sirt6显著抑制了促炎细胞因子的分泌,促进了炎症条件下BMSCs的成骨分化。

关键词: 骨髓间充质干细胞, 脂多糖, 骨生成, 沉默信息调控因子6

Abstract:

Objective: To investigate the effect of silent information regulator 6(Sirt6) on the bone formation of bone marrow mesenchymal stem cells(BMSCs) in the inflammatory microenvironment. Methods: Lipopolysaccharide(LPS) was used to induce the inflammatory reaction of primary BMSCs grown in adherent culture; the secretion of the proinflammatory cyto-kines including tumor necrosis factor-α(TNF-α), interleukin-1β(IL-1β) and IL-6 was analyzed by enzyme-linked immunosorbent assay(ELISA). The experiment included three groups, namely LPS group, LPS+blank group, and LPS+Sirt6 overexpression group. The mRNA expression levels of Sirt6 in three groups were analyzed respectively by real-time quantitative polymerase chain reaction(RT-qPCR) and protein levels of Sirt6 were analyzed by Western blot in inflammation settings. Mineralization of inflamed BMSCs in all groups was evaluated by alizarin red staining. Results: LPS could trigger inflammatory responses of the cultured BMSCs; the levels of TNF-α, IL-1β, and IL-6 were increased significantly(P<0.05); a plasmid vector was successfully prepared for overexpression of Sirt6 and transfected with the BMSCs; RT-qPCR and Western blot results showed that the mRNA and protein expression levels of Sirt6 in the LPS+Sirt6 overexpression group were significantly higher than those in the LPS group and LPS+blank group, while the expression levels of the osteogenic markers osteocalcin(OCN), alkaline phosphatase(ALP), bone sialoprotein(BSP) were relatively high in the LPS+Sirt6 overexpression group; the alizarin red staining demonstrated that the LPS+Sirt6 overexpression group had an increased number of calcified nodules. Conclusion: Overexpressed Sirt6 can significantly suppress the secretion of proinflammatory cytokines and promote osteoblastic differentiation of the inflamed BMSCs.

Key words: bone marrow mesenchymal stem cells, lipopolysaccharide, bone formation, silent information regulator 6

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