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目的构建新西兰兔骨髓间充质干细胞(bone mesenchymal stem cells,BMSCs)-庆大霉素-藻酸钙三维缓释培养体系,研究BMSCs的生长和分化情况。方法分别构建BMSCs-藻酸钙三维培养体系(W组)及BMSCs-庆大霉素-藻酸钙三维缓释培养体系(U组),在37℃、饱和湿度、5%CO_2培养箱内培养,培养液为高糖DMEM(含15%胎牛血清、10ng/mL转化生长因子β_1),隔日换液,观察细胞形态变化及凝珠形态变化。第2、4、6周对凝珠进行H-E染色、甲苯胺蓝染色及Ⅱ型胶原染色。结果两组三维凝珠培养第10天局部有细胞团簇形成;第21天可见大量细胞团簇形成,BMSCs多保持圆球形或类球形。两组细胞增殖及生长能力差异无统计学意义(P>0.05)。培养2周,两组凝珠甲苯胺蓝染色均呈阳性,但无明显细胞外基质形成,Ⅱ型胶原抗体染色均呈弱阳性;培养4周,两组凝珠甲苯胺蓝染色均显示位于细胞团块外周的细胞呈蓝色,中央的细胞染色不明显,细胞团外周蓝色染色物质较少,中央有较多紫红色物质,Ⅱ型胶原抗体染色均呈强阳性。H-E染色、甲苯胺蓝染色及Ⅱ型胶原抗体染色均未发现两组BMSCs的转化生长有差异,增殖数量差异也无统计学意义。结论适量的庆大霉素局部缓释并未明显影响BMSCs的生长及转化,又可达到最小抑菌浓度;庆大霉素对BMSCs及软骨细胞超微结构的影响有待进一步探讨。
Objective To construct New Zealand rabbit bone marrow mesenchymal stem cells (BMSCs) - gentamicin - calcium alginate three - dimensional sustained - release culture system to study the growth and differentiation of BMSCs. Methods BMSCs-calcium alginate three-dimensional culture system (W group) and BMSCs-gentamycin-calcium alginate three-dimensional sustained-release culture system were established respectively and cultured in a humidified atmosphere at 37 ℃ with 5% CO 2 . The culture medium was high glucose DMEM (containing 15% fetal bovine serum and 10ng / mL transforming growth factor β 1). The fluid was changed every other day. The morphological changes of the cells and the shape of the beads were observed. The beads were subjected to H-E staining, toluidine blue staining and type II collagen staining at the 2nd, 4th and 6th weeks. Results Two groups of three-dimensional dendritic beads were cultured on the 10th day with local cell clusters. On the 21st day, a large number of cell clusters were found, and the BMSCs remained spherical or spheroidal. There was no significant difference in cell proliferation and growth between the two groups (P> 0.05). After two weeks of incubation, the staining of toluidine blue in both groups showed positive staining, but no obvious extracellular matrix was formed. The staining of type Ⅱ collagen antibody was weakly positive. After 4 weeks of incubation, The cells in the periphery of the clumps were blue, the cells in the center were not obviously stained, there were fewer blue stained materials in the periphery of the clumps, more purple substances in the center, and strong positive staining for type Ⅱ collagen antibodies. H-E staining, toluidine blue staining and collagen type II staining did not find any difference in the number of transformed BMSCs between the two groups. There was also no significant difference in the number of proliferation between the two groups. Conclusion The moderate amount of gentamicin local slow release did not significantly affect the growth and transformation of BMSCs, but also reached the minimum inhibitory concentration. The effect of gentamicin on the ultrastructure of BMSCs and chondrocytes needs to be further explored.