Shh信号通路抑制剂Jervine对骨髓增生异常综合征MUTZ-1细胞的作用

Effect of Sonic Hedgehog Signal Pathway Inhibitor Jervine on Myelodysplastic Syndromes MUTZ-1 Cells

目的:研究Shh信号通路中SMO抑制剂(Jervine)对骨髓增生异常综合征MUTZ-1细胞株的增殖、凋亡和细胞周期的影响及可能的作用机制。方法:应用CCK-8法检测Jervine对MUTZ-1细胞增殖的影响;Annexin V/PI双染色流式细胞术检测Jervine对MUTZ-1细胞凋亡及细胞周期的作用;Western blot法检测Shh信号通路效应蛋白BCL2和CyclinD1的表达水平;荧光实时定量PCR法(RT-qPCR)检测Jervine作用于MUTZ-1细胞后Shh通路中Smo和Gli1基因的表达。结果:不同浓度Jervine对MUTZ-1细胞生长具有抑制作用,且抑制率呈剂量依赖性增加(24 h,r=-0.977);Jervine能有效促进细胞凋亡,并且细胞凋亡率与Jervine剂量成呈相关(r=0.997);加入不同浓度Jervine后G1期细胞所占比重增多,S期细胞减少(P<0.001),Jervine可使MUTZ-1细胞周期阻滞于G1期,抑制细胞增殖。Jervine作用MUTZ-1细胞后Smo和Gli1基因表达水平及下游靶基因的BCL2和CyclinD1蛋白表达水平都明显下降(P<0.05)。结论:SMO抑制剂能有效抑制MUTZ-1细胞增殖,促进其凋亡,其机制可能与下调BCL2和CyclinD1蛋白表达水平相关。

Objective:To study the effect of SMO inhibitor(Jervine)on proliferation,apoptosis and cell cycle of MDS cell line MUTZ-1,and its mechanism.Methods:The effect of different concentrations Jervine on proliferation of MUTZ-1 cells was detected by CCK-8 method.Apoptosis and cell cycle of MUTZ-1 cells were detected by flow cytometry.Western blot was used to detect the changes of Shh signaling pathway effecting proteins BCL2 and CyclinD1.The expression levels of Smo and Gli1 gene were detected by real-time fluorescent quantitative polymerase chain reaction(RTqPCR).Results:Jervine inhibited MUTZ-1 cell proliferation in a concentration dependent manner(24 h,r=-0.977),the apoptosis rate of MUTZ-1 cells increased with the enhancement of concentration of Jervine in MUTZ-1 cells(P<0.001),the cell proportion of G1 phase increased and the cell number of S phase decreased with enhancement of concentration(P<0.001).The result of RT-qPCR and Western blot showed that the expression of Smo,Gli1 mRNA and BCL2,CyclinD1 proteins decreased(P<0.05).Conclusion:SMO inhibitor can effectively inhibit the growth of MDS cell line MUTZ-1 improve the cell apoptosis and induce cell cycle arrest.Its action mechanism may be related with dowm-regulating the expression of BCL2 and CyclinD1.