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目的初步探讨Reversine对小鼠C2C12成肌细胞增殖分化的影响。方法体外培养C2C12成肌细胞系.实验第一步分三组:A组:正常对照组,B组:1μM Reversine处理12h组,C组:1μM Reversine处理24h组。上述三组用Annexin-V/PI双染法处理C2C12成肌细胞并用流式细胞仪技术检测三组C2C12细胞凋亡的影响;实验第二步分五组:D组:正常对照组,E组:单独1μM Reversine处理7d,F组:单独成骨诱导7d,G组:1μM Reversine诱导12h+单独成骨诱导7d,H组:1μM Reversine诱导12h+1μM Reversine联合成骨诱导7d。上述五组光镜下观察细胞形态的变化,并通过逆转录聚合酶链反应(RT-PCR)法检测五组C2C12成肌细胞分化抑制因子(ID2),肌肉发生调节因子(Myogenin),生肌因子后结蛋白(Desmin)mRNA的表达。结果与A组相比,C组1μM Reversine处理24h能引起C2C12细胞明显的凋亡,B组1μM Reversine处理12h的C2C12细胞未见明显的凋亡。与D组相比,E组和H组的细胞增殖明显受到抑制,F组和G组细胞逐渐增殖并融合。结论 Reversine能够显著的抑制成肌细胞的增殖分化过程。
Objective To investigate the effect of Reversine on proliferation and differentiation of mouse C2C12 myoblasts. Methods The C2C12 myoblast cell line was cultured in vitro.The first step was divided into three groups: group A: normal control group, group B: 1μM Reversine for 12h, group C: 1μM Reversine for 24h. The above three groups were treated with Annexin-V / PI double staining C2C12 myoblasts and flow cytometry was used to detect the apoptosis of three groups of C2C12 cells; the second step of the experiment divided into five groups: D group: normal control group, E group : Repetine treated with 1μM Reversine alone for 7d, Group F: 7d after osteogenic induction alone, Group G: Reversine induced by 1μM for 12h and induced by osteoblasts alone for 7d; Group H: Reversine induced by 1μM Reversine for 12h with 1μM Reversine for 7 days. The morphological changes of the cells were observed under the above five groups of light microscopes, and five groups of C2C12 myoblast differentiation inhibitor (ID2), myogenin, myogenic Factor Desmin mRNA expression. Results Compared with group A, C treated with 1μM Reversine for 24 hours caused obvious apoptosis in C2C12 cells, and no obvious apoptosis was observed in group B treated with 1μM Reversine for 12h. Compared with group D, the cell proliferation was obviously inhibited in group E and group H, and the cells in group F and group G gradually proliferated and fused. Conclusion Reversine can significantly inhibit the proliferation and differentiation of myoblasts.