具有成骨、成血管特性的三维细胞多聚体的异位成骨实验研究
首发时间:2019-05-07
摘要:目的:探讨利用兔骨髓间充质干细胞, 诱导形成的成骨细胞以及人脐静脉内皮细胞体外共培养形成的三维微组织的体内异位成骨能力。方法:体外分离培养兔骨髓间充质干细胞(Bone marrow derived mesenchymal stem cells, BMSCs),诱导分化为成骨细胞,并与人脐静脉内皮细胞在低黏附培养板中共培养,形成三维细胞多聚体,对照组为干细胞与诱导的成骨细胞共培养,植入裸鼠皮下,分别在2周及4周用碱性磷酸酶、茜素红染色检测植入组织的成骨能力,用CD31免疫荧光染色检测成血管能力。结果:兔骨髓间充质干细胞分离培养后生长活性高,细胞呈长梭形,旋涡状生长,向成骨方向诱导分化两周后,细胞呈多角形或立方形,表面有短突起,茜素红染色见橙红色钙结节。在96孔低黏附培养板中培养1周,肉眼观察可见细胞聚集成球状,茜素红染色及碱性磷酸酶染色表明两组微球均有成骨能力,CD31免疫荧光显示共培养组有成血管潜力。裸鼠皮下植入2周,OB组与共培养组植入位点均发现组织结节,4周后,两组植入位点处组织结节变小,边界清晰,表面按压较硬。茜素红染色发现共培养组较OB组橙红色钙结节多,HE染色发现共培养组形成血管数目远多于OB组,差异均具有统计学意义。结论:用骨髓间充质干细胞、成骨细胞及人脐静脉内皮细胞体外构建的三维微组织植入裸鼠体内后具有成骨成血管的潜能。
关键词: 细胞生物学 兔骨髓间充质干细胞 低黏附培养 三维微组织 异位成骨
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The research about Ectopic osteogenesis of 3-D cell spheres with osteogenesis and angiogenic properties
Abstract:Objective: To investigatethe ectopic osteogenic capacity of the micro-tissue constructed with rabbit bone marrow mesenchymal stem cells, osteoblast cells and human umbilical vein endothelial cells in vivo. Method: Rabbit bone marrow mesenchymal stem cells (BMSCs) are isolated and inducted to osteoblasts; then co-cultured with human umbilical vein endothelial cells to construct three-dimensional spheres in the low-adhesion culture plate. BMSCs and osteoblasts are co-cultured in the control group.after 7 days Spheres are cultured in vitro, they aretransplanted into nude mice. The osteogenic capacity and angiogenic ability of the implanted tissues were detected at 2nd and 4th weeks respectively with ALP staining and alizarin red staining, the angiogenic capacity detected with CD31 immunofluorescence. Results: Rabbit bone marrow mesenchymal stem cells were isolated and cultured, which were long fusiform and spirally growing,After induced into osteogenic direction for two weeks, the cells became polygonal or cubic, with short protrusions on the surface, and orange-red calcium nodules were stained with alizarin red. After cells were cultured in 96-well low-adhesion culture plates for 1 week, the cells were observed to be spheroidized by naked eyes. The alizarin red staining and alkaline phosphatase staining showed that both groups of microspheres had osteogenesis. The CD31 immunofluorescence showed an angiogenic potential in the co-culturing three kinds of cells group. After two weeks of implantation, tissue nodules were found in the OB group and the co-culture group. After 4 weeks, the tissue nodules at the implantation sites became smaller with clear boundary and the surface compression was hard. Alizarin red staining showed that the co-culture group formed more bone than OB. HE staining showed that the number of blood vessels formed in the co-culture group was much higher than that in the OB group. Conclusion: Three-dimensional micro-tissue constructed with bone marrow mesenchymal stem cells, osteoblasts and human umbilical vein endothelial cells in vitro has the capacity of osteogenesis and angiopoiesis
Keywords: cellbiology rabbit bone marrow mesenchymal stem cells low-adhesion culture 3D micro-tissue ectopic osteogenesis
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具有成骨、成血管特性的三维细胞多聚体的异位成骨实验研究
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