The University of Melbourne, Department of Medicine, and Cooperative Research Centre for Chronic Inflammatory Diseases, Royal Melbourne Hospital, Parkville, Victoria, Australia.
Osteoarthritis Cartilage. 2009 Jun;17(6):735-42. doi: 10.1016/j.joca.2008.11.011. Epub 2008 Nov 24.
The effects of inflammation on bone development from mesenchymal stem cells (MSC) are unclear due to the difficulty in isolating MSC. The aim of this study was to develop a MSC isolation method and to determine the in vitro effects of interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNFalpha) on their osteogenic differentiation.
Murine MSC were isolated from the limbs of C57/Bl6 mice through collagenase digestion of bone and enriched as the Stem cell antigen (Sca-1)(+) CD31(-) CD45(-) population, using lineage immunodepletion, followed by fluorescence-activated cell sorting (FACS). They were differentiated along the osteoblast linage in the presence or absence of IL-1beta and TNFalpha. Mineralization was measured as was the expression of a number of osteogenic genes by quantitative polymerase chain reaction (PCR).
We show that osteogenic differentiation from the MSC population is suppressed by IL-1beta and TNFalpha. In addition to suppression of bone mineralization, both cytokines inhibited the differentiation-associated increases in alkaline phosphatase (ALP) activity and the gene expression for ALP, alpha1(I) procollagen, runt-related transcription factor 2 (Runx2) and osterix. However, only TNFalpha inhibited osteonectin and osteopontin mRNA expression and only IL-1beta reduced cell proliferation.
The convenient isolation technique enables the easy generation of sufficient MSC to permit the molecular analysis of their differentiation. We were thus able to show that the proinflammatory cytokines, IL-1beta and TNFalpha, can compromise bone development from this primary MSC population, although with some significant differences. The potential involvement of specific inflammatory mediators needs to be taken into account if optimal bone repair and presumably that of other tissues are to be achieved with MSC.
由于间充质干细胞 (MSC) 的分离困难，炎症对其成骨发育的影响尚不清楚。本研究旨在开发一种 MSC 分离方法，并确定白细胞介素-1β (IL-1β) 和肿瘤坏死因子 α (TNFα) 对其成骨分化的体外影响。
通过胶原酶消化骨骼从 C57/Bl6 小鼠的四肢中分离出鼠 MSC，并用谱系免疫耗竭法和荧光激活细胞分选 (FACS) 将其富集为干细胞抗原 (Sca-1)(+) CD31(-) CD45(-) 群体。在存在或不存在 IL-1β 和 TNFα 的情况下，它们沿着成骨细胞谱系分化。通过定量聚合酶链反应 (PCR) 测量矿化和许多成骨基因的表达。
我们表明，MSC 群体的成骨分化受到 IL-1β 和 TNFα 的抑制。除了抑制骨矿化外，两种细胞因子还抑制了与分化相关的碱性磷酸酶 (ALP) 活性和 ALP、α1(I) 前胶原、转录因子 2 (Runx2) 和骨桥蛋白基因表达的增加。然而，只有 TNFα 抑制了骨粘连蛋白和骨桥蛋白 mRNA 的表达，只有 IL-1β 降低了细胞增殖。
方便的分离技术使我们能够轻松生成足够的 MSC，从而对其分化进行分子分析。因此，我们能够表明，促炎细胞因子 IL-1β 和 TNFα 可损害这种原发性 MSC 群体的骨发育，尽管存在一些显着差异。如果要通过 MSC 实现最佳的骨修复和推测的其他组织修复，则需要考虑特定炎症介质的潜在参与。
