摘要
目的探讨大鼠骨髓间充质干细胞(BM-MSCs)培养换液模式。方法全骨髓贴壁法培养SD大鼠BM-MSCs,按首次换液方式分为以下6组:A组24h半量换液;B组24h全量换液;C组48h半量换液;D组48h全量换液;E组72h半量换液;F组72h全量换液,以后各组每48h全量换液。记录各组第0代至第3代(P0～P3)培养时间及细胞纯度,通过传代培养、扩增,倒置光学显微镜及原子力显微镜下观察细胞形态;MTT法检测细胞增殖曲线,流式细胞仪检测免疫表型;体外向成骨、脂肪方向诱导分化,并通过特异性染色鉴定其分化能力。结果各组培养总时间比较有统计学意义(F=46.455,P<0.05),D组最短(16.61±1.61)d,B组最长(21.50±1.70)d,B、D组与各组间差异均有统计学意义(P<0.05)。B组各代纯度均最高,P3纯度达(90.84±2.19)﹪。E组各代细胞纯度均为最低,P3纯度为(76.04±3.21)﹪。P3纯度比较,B、E组与其他各组差异有统计学意义(P<0.05)。所获细胞CD29、CD90阳性,CD34、CD45阴性,向成骨、成脂诱导分化,MTT检测显示细胞增殖能力良好。结论首次换液方式能影响大鼠BM-MSCs的培养周期及其纯度,48h半量换液为最佳首次换液方式。
Objective To study the influence of different modalities of the first medium exchange on culturing bone marrow-derived mesenchymal stem cells of rat by attachment method.Methods On condition of modality of the first medium exchange,6 experimental groups were performed as follows:group A was half exchanged after 24?h,group B was all exchanged after 24?h,group C was half exchanged after 48 hours,group D was all exchanged after 48?h,group E was half exchanged after 72?h,group F was all exchanged after 72?h.Then,all groups were all exchanged every 48?h and were subcultured consecutively.The period and purity of each passage were measured.Cells of P4 were identified by flow cytometry,osteoblasts and adipocytes differentiation and measured proliferatal ability by MTT method.Results The total culture time was significant different(F?=?46.455,P?<?0.001).Group D(16.61?±?1.61)d was the least while group B(21.50?±?1.70)d longest,and both of the two groups were significant different compared with others.Group B earned the highest purity in all passages with(90.84?±?2.19)﹪ of P3 and group E the lowest(76.04 ±?3.21)﹪,and both of the two groups were significant different compared with others.Cells of P4 showed CD29、CD90 positive and CD34、CD45 negative by flow cytometry test,induced to differentiate into osteoblasts and adipocytes and were in good proliferation conditation by MTT.Conclusion The modality of the first medium exchange had an influence on the culture period and purity of BM-MSCs,and the modality of 48h half exchange was the best protocol.
出处
《功能与分子医学影像学（电子版）》
2012年第2期97-102,共6页
Functional and Molecular Medical Imaging(Electronic Edition)
基金
南京军区医学科技创新重点课题(09Z029)
南京军区重点课题(10Z030)
关键词
换液
贴壁分离
纯度
周期
骨髓间充质干细胞
the first medium exchange
attachment method
purity
period
bone marrow-derived mesenchymal stem cells
