骨髓间充质干细胞机械刺激成骨的记忆效应

Memory effect of bone marrow mesenchymal stem cells on osteogenesis after mechanical stimulation

背景:骨髓间充质干细胞作为组织工程的理想种子细胞,已证实力学刺激有引导其分化的作用。研究表明,干细胞存在着机械记忆,即力学刺激所引起的改变可能会长期影响干细胞的命运。目的:观察骨髓间充质干细胞对力学刺激的记忆效应,解析其发生的表观遗传学机制。方法:(1)分离原代小鼠骨髓间充质干细胞,检测其三系分化能力,传至第3代待用;(2)利用拉伸仪,体外给予小鼠骨髓间充质干细胞机械刺激,检测细胞拉伸前后的成骨分化情况;(3)流式细胞仪检测拉伸后细胞干性;(4)将拉伸处理与未处理的细胞消化后铺种在常规12孔板,继续培养14 d后检测细胞的成骨分化情况;(5)免疫荧光染色检测并筛选造成干细胞机械记忆的关键组蛋白修饰变化,初步明确表观遗传调控的可能机制。结果与结论:(1)5%形变,0.5 Hz,6 h/d,为期7 d的机械刺激可以引导小鼠骨髓间充质干细胞向成骨方向分化;(2)重新铺种培养皿后,拉伸处理后的细胞仍能保持干性且继续向成骨方向分化;(3)结果显示,骨髓间充质干细胞对机械刺激存在记忆效应,这种效应可能与力学刺激导致的组蛋白H3K4me3修饰有关。

BACKGROUND:Bone marrow mesenchymal stem cells are ideal seed cells for tissue engineering,and mechanical stimulation can guide their differentiation.It has been found that stem cells have mechanical memory,that is,the change induced by mechanical stimulation may affect the long-term fate of stem cells.OBJECTIVE:To investigate the memory effect of bone marrow mesenchymal stem cells on mechanical stimulation and to analyze the epigenetic mechanism.METHODS:(1) Mouse bone marrow mesenchymal stem cells were isolated,and the differentiation ability of the three-lineage was detected for further use at passage 3.(2) Mouse bone marrow mesenchymal stem cells were stimulated using extensometer in vitro to detect osteogenic differentiation before and after stretching.(3) The stemness of cells after stretching was detected by flow cytometry.(4) The stretched and unstretched cells were re-digested into ordinary 12-well plates and cultured for 14 days to detect the osteogenic differentiation of the cells.(5) Immunofluorescence staining was used to detect and screen the key histone modification changes that cause the mechanical memory of stem cells,and to preliminarily clarify the possible mechanism of epigenetic regulation.RESULTS AND CONCLUSION:(1) Mechanical stimulation of 5% deformation,0.5 Hz,6 hours per day for 7 days could induce the differentiation of mouse bone marrow mesenchymal stem cells to osteogenic direction.(2) After reseeding plate culture,the stretched cells remained dry and continued to differentiate toward osteogenesis.(3) The results showed that bone marrow mesenchymal stem cells had epigenetic memory for mechanical stimulation,and this epigenetic memory may be related to histone H3 K4 me3 modification,which is induced by mechanical stimulation.