摘要
背景:传统的细胞培养方法大多采用胎牛血清作为营养支持,其存在传播疾病的风险和免疫排斥反应。寻找胎牛血清替代物并建立规范、安全、高效的成人骨髓基质干细胞体外培养体系刚刚起步,有待进一步研究。目的:探讨AB血清、自体血清替代胎牛血清体外培养人骨髓基质干细胞的效果。方法:从成人骨髓穿刺液中分离人骨髓基质干细胞,分别用含体积分数10%AB血清、自体血清和胎牛血清的基础培养基培养人骨髓基质干细胞,取第3代细胞进行相关指标检测。结果与结论:相差显微镜下见AB血清组细胞最先达到汇合;流式细胞仪检测细胞表面抗原,各组均符合骨髓干细胞表型,纯化程度良好;Alamar Blue法显示AB血清组平均荧光强度最高,促增殖效率最高;Annexin V-FITC法显示各组细胞凋亡率均在5%以下,生长状态良好;碱性磷酸酶染色、钙结节和油红O染色结果显示,各组细胞均保持了成骨、成脂分化能力。AB血清对人骨髓基质干细胞的增殖作用较胎牛血清、自体血清明显增强。AB血清有望替代胎牛血清建立符合骨组织工程临床应用要求的体外成人骨髓基质干细胞培养体系。
BACKGROUND： Fetal bovine serum as nutritional support is often used in the traditional cell culture. Consequently, a host of potential problems such as the spread of disease and immunological reactions exist. To find a suitable fetal bovine serum substitute and to establish a culture system of human bone marrow stromal stem cells in vitro which has been standardized, safe and efficient has just started.OBJECTIVE： To investigate the effects of different serums on proliferation of bone marrow stromal stem cells in vitro. METHODS： Bone marrow stromal stem cells were obtained from adult bone marrow, which were cultured in DMEM containing 10% AB serum, 10% autologous serum, or 10% fetal bovine serum. Cells at passage 3 were used in this study.RESULTS AND CONCLUSION： The cell confluence in the AB serum group was earlier than that in the fetal bovine serum group and autologous serum group. Human bone marrow stromal stem cells maintained the phenotypes of bone marrow stem cells in three serums detected by flow cytometry. AB serum group showed the highest fluorescence intensity and the most efficiency of cell proliferation which examined by the AlamarB lue assay. Apoptosis rate was 5% in all the three groups, and cells grew well in these serums. Alkaline phosphatase, calcium nodules and oil red O staining showed that the cells maintained the osteogenesis and adipogenesis capacity in the three groups. AB serum was found to have a better effect on proliferation capability of cells than fetal bovine serum and autologous serum. Taken together, AB serum is expected to be a substitute of fetal bovine serum to build an in vitro culture system of adult bone marrow stromal stem cells that accord with the clinical requirements of bone tissue engineering.
出处
《中国组织工程研究》
CAS
北大核心
2015年第45期7242-7248,共7页
Chinese Journal of Tissue Engineering Research
基金
国家自然科学基金项目(31470937
331300798)
南方医科大学南方医院院长基金项目(2014CO14)~~
