目的 分析不同处理条件下细胞同步化于G0/G1时期的效果以得到最佳的同步化条件，并研究同步化对骨髓间充质干细胞(BMSCs)在碱性成纤维细胞因子(bFGF)的作用下分化为神经细胞的影响。方法 分离培养成年大鼠BMSCs，5%、1%、0.5%、0.1%、0胎牛血清(FBS)分别处理24 h、48 h。PI染色后经流式细胞仪检测细胞周期各时相细胞所占比例，与正常培养条件(10% FBS)处理下对比。得到最佳处理条件后，bFGF处理3 d、7 d，免疫荧光细胞化学方法检测Nestin和Tuj-1的表达。结果 成年大鼠BMSCs原代提取，经传代后，细胞形态为长梭形。不同处理条件下G1/G0期细胞比例均高于正常培养条件，1% FBS处理48 h时G1/G0期细胞比例为(94.274±0.468)%，达最高峰(F=39.91, P<0.001)。bFGF诱导3 d后，细胞周期同步化后的Nestin+细胞数显著高于未同步化的细胞数[(80.3±2.4)% vs. (12.1±1.5)%] (F=28.25, P<0.001)；bFGF诱导7 d后，同步化后的Tuj-1+细胞数显著高于未同步化的细胞数[(74.8±3.2)% vs. (19.3±2.5)%] (F=17.95, P<0.001)。结论 1% FBS处理48 h是将BMSCs同步化于G0/G1期的最佳条件。细胞周期同步化于G0/G1期能够提高BMSCs向神经细胞分化的比例。
Objective To analyze the effect of different treatment conditions on cells synchronization in G0/G1 phase to get the best condition, and to explore its effect on neural differentiation of bone marrow mesenchymal stem cells (BMSCs) induced by basic fibroblast growth factor (bFGF).Methods BMSCs were isolated and cultured in 5%, 1%, 0.5%, 0.1%, 0 fetal bovine serum (FBS) respectively, for 24 hours and 48 hours. After PI staining, cell cycle proportions of each phase were detected by flow cytometry, and were compared with the normal group (10% FBS). After the optimal treatment condition was got, 20 ng/ml bFGF was added into synchronization group and unsynchronization group 3 days and 7 days, respectively. The expression of Nestin and Tuj-1 were detected with immunofluorescence.Results Adult rat BMSCs were isolated from bone marrow and cultured, after passage, the cells were with long spindle shape. Compared with the normal group, the cell proportion of G1/G0 phase increased under different treatments, peaked with (94.274±0.468)% under 1% FBS, 48 hours (F=39.91, P<0.001). After bFGF induction for 3 days, the Nestin+ cell number was higher in the synchronization group than in the unsynchronization group [(80.3±2.4)% vs. (12.1±1.5)%] (F=28.25, P<0.001). After bFGF induction for 7 days, the Tuj-1+ cell number was higher in the synchronization group than in the unsynchronization group [(74.8±3.2%)% vs. (19.3±2.5)%] (F=17.95, P<0.001).Conclusion 1% FBS, 48 hours is the optimal condition to BMSCs synchronization in G0/G1 phase, which can promote the neural differentiation of BMSCs.
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