Reversine对小鼠C2C12成肌细胞增殖与分化作用的初步研究

The proliferation and differentiation of C2C12 cells treated with reversine in vitro

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 effects of reversine on the proliferation and differentiation of C2C12 myoblasts in vitro.Methods C2C12 cells cultured in vitro were treated with 1μM reversine about 12h and 24h respectively,and then flow cytometric analysis was used to detect the apoptosis of treated cells after staining with Annexin-V/PI.Furthermore,myoblast were divided into five groups：control group（CG）,reversine group（Revs only）,bone induction factor group（BIF only）,reversine and bone induction factor group（Revs and BIF）,reversine,bone induction factor and reversine group（Revs and BIF and Revs）.The morphological changes of myoblast were observed under the inverted microscope.The mRNA expression of inhibitor of differentiation（ID2）,muscle regulatory factor（Myogenin） and myogenic factor（Desmin） was detected by reverse transcription-polymerase chain reaction（RT-PCR） respectively.Results：Compared with group A,significant apoptosis of C2C12 cells appeared in those treated with 1μM reversine about 24h,whereas,no significant apoptosis appeared in group B.Compared with group D,proliferation was significantly inhibited in cells of group E and H,however,cell proliferated and differentiated（fusion） in group F and G.Conclusions Reversine significantly inhibits the proliferation and differentiation of cultured myoblasts.