SDF-1α复合Pluronic F-127对BMSCs体外诱导成骨的实验观察

发布时间：2018-08-17 09:23

【摘要】：目的以Pluronic F-127为支架材料，构建装载基质细胞衍生因子-1α（stromal cellderived factor-1α，SDF-1α）的Pluronic F-127新型复合材料，探讨其对大鼠骨髓间充质干细胞（bone marrow stromal cells，BMSCs）的体外趋化作用，并将PluronicF-127与BMSCs复合培养，观察两者间生物相容性，进而研究复合SDF-1α水凝胶对BMSCs诱导分化成成骨细胞的影响。研究方法构建装载不同浓度SDF-1α（200、50ng/ml）的Pluronic F-127复合水凝胶，采用Transwell小室法进行复合水凝胶对BMSCs的体外趋化实验，通过对迁移BMSCs的计数与统计分析，筛选出趋化作用较好的复合水凝胶。将BMSCs与筛选出的复合水凝胶均匀混合培养，用MTT法检测复合培养后细胞的活性状况。将BMSCs与筛选出的复合水凝胶混合均匀，待凝胶形成后加成骨诱导液进行培养，同时分别设置单纯BMSCs的成骨诱导液培养和完全培养基培养，对以上各行碱性磷酸酶（alkaline phosphatase，AKP）活性测定及茜素红染色，观察并记录各检测结果，分析各BMSCs的成骨诱导分化情况。结果趋化结果显示，含SDF-1α浓度为200ng/ml的复合水凝胶对BMSCs的募集效果显著，与含SDF-1α浓度为50ng/ml的复合水凝胶相比，其趋化下室滤膜表面的细胞转移数目明显增多（P0.05）。MTT法证实Pluronic F-127对细胞增殖不产生影响，BMSCs-水凝胶复合培养与单纯BMSCs培养相比，两者间细胞活性差异并无统计学意义（P0.05）。AKP活性测定表明，，BMSCs-复合水凝胶、单纯BMSCs在成骨诱导培养条件下，两者中细胞的AKP表达量均明显增高，并且都高于非成骨诱导组（P0.05），同时经成骨诱导液培养的细胞，在镜下均可见明显的橘红色钙结节团块，而非加成骨诱导液培养的细胞，镜下则未见钙结节形成。结论成功构建装载SDF-1α的Pluronic F-127新型复合材料，其在体外对BMSCs有明显的趋化作用，在Pluronic F-127水凝胶中，BMSCs能够存活较好并能进行正常的生长及增殖，并且在细胞-复合水凝胶复合培养的三维支架中，BMSCs能够被成功诱导分化为成骨细胞。
[Abstract]:Objective to construct a novel Pluronic F-127 composite loaded with stromal cell derived factor 1 伪 (stromal cellderived factor-1 伪 (SDF-1 伪) and investigate its chemotactic effect on rat bone marrow mesenchymal stem cells (bone marrow stromal cells) in vitro, and to co-culture PluronicF-127 with BMSCs. The effect of SDF-1 伪 hydrogel on the differentiation of osteoblasts induced by BMSCs was studied. Methods Pluronic F-127 hydrogels loaded with different concentrations of SDF-1 伪 (200ng / ml) were constructed. The chemotactic experiments of BMSCs were carried out by Transwell chamber method. The chemotactic complex hydrogels with good chemotaxis were screened by counting and statistical analysis of BMSCs migration. The BMSCs and the selected composite hydrogels were cultured uniformly and the activity of the cells was detected by MTT method. The mixture of BMSCs and the selected composite hydrogels was mixed evenly. After the gel was formed, the osteoblast was cultured with osteogenic inducer, and the pure BMSCs osteogenic medium and the complete culture medium were set up respectively. The activity of alkaline phosphatase (alkaline) and alizarin red staining were measured, the results were observed and recorded, and the osteogenic differentiation of each BMSCs was analyzed. Results the chemotaxis results showed that the composite hydrogel containing SDF-1 伪 concentration of 200ng/ml had significant effect on the recruitment of BMSCs, compared with the composite hydrogel containing SDF-1 伪 concentration of 50ng/ml. The number of cell metastases on the surface of the membrane increased significantly (P0.05). The results showed that Pluronic F-127 had no effect on cell proliferation. Compared with BMSCs culture, Pluronic F-127 had no effect on cell proliferation. There was no significant difference in cell activity between the two groups (P0.05) .AKP activity test showed that the expression of AKP was significantly increased in osteogenic culture with BMSCs alone. Both of them were higher than those of non-osteogenic induction group (P0.05). At the same time, the cells cultured with osteoblast could be seen the obvious orange calcium nodule mass under the microscope, but the cells cultured with non-additive osteoinductive fluid had no calcium nodule formation under the microscope. Conclusion the novel Pluronic F-127 composite loaded with SDF-1 伪 has obvious chemotactic effect on BMSCs in vitro. It can survive well and can grow and proliferate normally in Pluronic F-127 hydrogel. BMSCs could be successfully induced to differentiate into osteoblasts in three dimensional scaffolds cultured with cell-composite hydrogel.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R783

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