吴燕丽，毕小军，宫晨，张明生.机械生长因子对骨髓间充质干细胞迁移作用的影响及相关机制研究[J].中华物理医学与康复杂志,2018,40(8):569-574
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| 机械生长因子对骨髓间充质干细胞迁移作用的影响及相关机制研究 |
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| DOI： |
| 中文关键词: 机械生长因子 骨髓间充质干细胞 迁移 RhoA 粘附斑 |
| 英文关键词: Mechano growth factor Mesenchymal stem cells Migration RhoA Adhesion |
| 基金项目:湖北省卫生计生科研基金（WJ2017M058） |
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| 中文摘要: |
| 目的 探讨机械生长因子（MGF）调控骨髓间充质干细胞（MSCs）迁移作用机制。 方法 分离Sprauge-Dawley(SD)大鼠的MSCs，外源性给予不同浓度（0，1，10，25，50 μM）MGF干预，采用Western Blot方法检测细胞骨架相关蛋白RhoA及其下游p-MYPT和粘附斑激酶相关蛋白p-FAK、t-FAK表达，探讨MGF对MSCs细胞骨架张力和粘附斑的调节作用；然后通过肉毒素C3抑制RhoA活性，采用Western Blot方法检测p-MYPT、p-FAK及FAK蛋白表达，采用Transwell跨膜小室实验检测细胞迁移能力，采用免疫荧光检测MSCs细胞骨架及粘附斑形成情况。 结果 MGF可明显促进MSCs RhoA激酶激活及其下游p-MYPT表达，且该效应具有浓度依赖性，以MGF浓度为50 μM时RhoA及p-MYPT表达尤为显著；p-FAK/t-FAK结果提示MGF可激活粘附斑激酶FAK，促进粘附斑形成；使用肉毒素C3抑制RhoA活性后发现FAK激活受到明显抑制；Transwell实验结果提示MGF可促进MSCs迁移，而经肉毒素C3干预后能明显抑制MSCs迁移；免疫荧光染色结果提示MGF可促进MSCs细胞骨架重排及粘附斑形成，而经肉毒素C3干预后可见细胞骨架破坏及粘附斑形成明显减少。 结论 MGF通过控制RhoA-FAK信号轴介导细胞骨架及粘附斑形成，从而调控MSCs迁移。 |
| 英文摘要: |
| Objective To study the mechanism by which mechano growth factor (MGF) promotes the migration of mesenchymal stem cells (MSCs). Methods MSCs were isolated from Sprague-Dawley rats and treated with MGF at various concentrations. Western blotting was used to evaluate the expression of RhoA protein and its downstream p-MYPT, as well as p-FAK and t-FAK proteins related to focal adhesion kinase. The aim was to illustrate the effect of MGF in regulating the cytoskeleton of MSCs and the formation of focal adhesion. C3 toxin was used to inhibit RhoA activity and western blotting was used to examine the expression of p-MYPT, and the focal adhesion kinases p-FAK and t-FAK. Transwell assays were used to examine MSCs′ migration ability, and immunofluorescence was conducted to examine the formation of F-actin cytoskeleton and focal adhesion. Results MGF can significantly promote the expression of MSCs′ RhoA and its downstream protein p-MYPT. The effect is dose-dependent. The expression of RhoA and p-MYPT increased most significantly at 50 μM concentration. The ratio of p-FAK to t-FAK indicates that MGF can activate focal adhesion kinase and promote adhesion. C3 toxin significantly inhibited FAK activation. Transwell assays showed that MGF can significantly promote MSC migration, but pretreatment with C3 toxin inhibited it. The immunofluorescence results show that MGF can promote the rearrangement of MSCs′ F-actin cytoskeleton and the formation of focal adhesion. C3 toxin disrupted MSCs cytoskeletons and decreased focal adhesion. Conclusion MGF promotes MSCs′ migration through RhoA- and kinase-mediated cytoskeleton rearrangement and the formation of focal adhesion. |
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