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

中图分类号: 

• R782