血清量及首次换液时间对荧光小鼠骨髓基质细胞体外增殖的影响

Effect of Different FSB Concentration and First-time Exchange of Total Volume Medium on Proliferation of Bone Marrow Stromal Cells from GFP Transgenic Mouse in vitro

目的研究不同量的血清及首次换液时间对荧光小鼠骨髓基质细胞(BMSCs)体外增殖的影响。方法转基因荧光小鼠BMSCs取材后种植于24孔培养板中,分别用含10%、20%、30%胎牛血清的DMEM/F12混合培养基常规培养。于4h、6h、8h、10h、12h、24h、48h、72h首次全量换液,观察其生长情况,用CD44、CD54、CD45抗体对10%、20%胎牛血清传代培养至第5代的细胞进行免疫细胞化学染色。结果BMSCs在含10%、20%胎牛血清的DMEM/F12中生长较好,原代培养的细胞其贴壁细胞数随首次换液时间的延长而增多,但纯度下降。首次换液时间相同而培养液血清含量不同的两组细胞免疫组织化学染色的阳性率差别无显著性(P>0.05),CD44和CD54抗体反应的阳性率随着首次换液时间的延长而逐渐降低,CD45抗体反应的阳性率则随首次换液时间的延长而逐渐升高。结论贴壁分离纯化对转基因荧光小鼠的BMSCs体外培养有效,其体外培养适宜的血清体积分数为0.1～0.2,首次换液时间以8h时为佳。

Objective To evluate the effect of different fetal bovine serum(FBS) concentration and first-time exchange of total volume medium on the proliferation of bone marrow stromal cells (BMSCs) from green fluorescence protein (GFP) transgenic mouse in vitro. Methods Bone marrow cells isolated from GFP transgenic mice were cultured in DMEM/F12 containing 10%, 20%, 30% FBS respectively; the first exchange of the total volume medium was made at different times(4 h, 6 h, 8 h, 10 h, 12 h, 24 h, 48 h and 72 h) after 3 d primary culture; then the total volume medicum was exchanged every three days. The amplification of BMSCs was determined. The passage 5 BMSCs cultured in DMEM/F12 containing 10% and 20% FBS were examined with the antibodies CD44, CD45 and CD54 at the time of first exchange of the total volume medium. Results The cultured cells proliferated well in DMEM/F12 containing 10% FBS and 20% FBS. With the extension of the time for first exchange of total volume medium, the density of the adhered cells increased, but the purity of BMSCs decreased. Conclusion The method of making cells adhere to culture plastic in different time for cultivating and purifying BMSCs from GFP transgenic mice is effective, the appropriate concentration of FBS is 10%-20% and the best time for the the first exchange of total volume medium is 8 hour.