摘要
目的:探讨抑制和激活Wnt信号通路在甾醇类新药NSC67657诱导HL-60细胞单核系分化中的作用和可能机制。方法:用5、10和20μmol/L的Wnt信号通路抑制剂XAV-939作用于HL-60细胞3 d,用10、20和30mmol/L的Wnt信号通路激活剂Li Cl作用于HL-60细胞1 d,应用RT-RCR和Western blot检测Wnt信号通路的下游靶点基因和蛋白表达水平,流式细胞术检测细胞表面分化抗原CD14的表达和细胞早期凋亡,筛选最佳抑制浓度和激活浓度。然后用10μmol/L NSC67657分别对已经抑制及激活了的Wnt通路的HL-60细胞作用3 d。分析比较空白对照组、NSC67657单独处理组和激活/抑制剂作用后NSC67657处理组的细胞表面抗原CD14和Wnt下游靶点蛋白的表达水平。结果:20μmol/L XAV-939和20 mmol/L Li Cl能分别有效抑制和激活HL-60细胞Wnt信号通路,分别显著下调和上调cyclin D1,TCF1,c-Jun基因(P<0.05)和蛋白(P<0.05)的表达,且在该条件下CD14^+HL-60细胞不超过1%,没有早期凋亡发生;NSC67657单独作用HL-60细胞能下调Wnt通路下游靶点cyclin D1、TCF1和c-Jun的蛋白表达(P<0.05),CD14^+细胞占(62.13±9.44)%;NSC67657作用于经XAV-939预处理的细胞,可使CD14^+细胞达到(84.17±5.39)%,cyclin D1,TCF1和c-Jun蛋白表达比单独NSC67657处理组降低更显著(P<0.01);而NSC67657作用经Li Cl预处理的细胞,可使CD14^+细胞减少至(33.99±8.37)%,cyclin D1,TCF1和c-Jun蛋白表达较非处理组下降明显较低(P<0.05),但高于单独使用NSC67657处理组(P<0.05)。结论:20μmol/L XAV-939和20 mmol/L Li Cl作为HL-60细胞Wnt信号通路的有效抑制剂和激活剂能够分别显著下调和上调Wnt下游通路靶点基因和蛋白表达,其干预后对NSC67657诱导细胞分化的影响提示Wnt信号通路可能在NSC67657诱导HL-60细胞单核系分化中起到关键作用。
Objective:To investigate the effect of inhibiting and activating Wnt signalling pathway on monocyte differentiation of HL-60 cells induced with a new steroidal drug NSC67657 and its possible mechamism.Methods:The HL-60 cells were treated with 5,10 and 20 μmol/L XAV-939(inhibitor of Wnt signalling pathway) for 3 days,and with 10,20 and 30 mmol/L LiCl(activator of Wnt signalling pathway) for 1 day;the expression levels of down-stream genes and proteins of Wnt signolling pathway were detected by RT-PCR and Western blot,respectively;the expression of cell surface differentiation antigen CD14 and early apoptosis of HL-60 cells was detected by flow cytometry,moreover the most suitable concentration of Wnt inhibitor and activator for HL-60 cells was determined.Then the HL-60 cells with inhibited and activated Wnt pathway were treated with NSC67657 of 10 μmol/L for 3 days;the expression levels of CD14 and down-stream target proteins of Wnt signalling pathway in blank control(culture mediam) group,simple NSC67657- treated group,NSC67657 combined with inhibitor group and NSC67657 combined activator group were compared and analyzed.Results:20 μmol/L XAV-939 and 20 mmol/L LiCl could effectively inhibit and activate Wnt signalling pathway of HL-60 cells respectively,could significantly down- and up-regulate the expression of cyclinDl,TCF1 and c-Jun genes(P <0.05) and proteins(P <0.05);moreover,the number of CD10^+ HL-60 cells in these conditions was below 1%,no early apoptosis of HL-60 cells was found.In the simple NSC67657-treated groups,the expression of cyclinDl,TCF1 and c-Jun proteins was down-regulated(P < 0.05),and the percentage of CD14^+ HL-60 cells accounted for 62.13 ±9.44;after the HL-60 cells were treated with XAV-939,the NSC67657 could more significantly down-regulate the expression of cyclinD1,TCF1 and c-Jun proteins and the percentage of CD14^+ HL-60 cell accounted for 84.17 ±5.39%,as compared with simple NSC67657- treated group;as compared with blank controls group,the expression of cyclinDl,TCF1 and c-Jun proteins was more obviously down-regulated and the percentage of CD14^+ HL-60 cells decreased to 33.99 ±8.37%in NSC67657 combined LiCl streated group,but which were higher than those in simple NSC67657-treated group(P < 0.05).Conclusion:20 μmol/L XAV-939 and 20 mmol/L LiCl as effective inhabitor and activator of Wnt signalling pathway respectively can significantly down- and up-regulate the expression of Wnt down-stream pathway target genes and proteins.The influence of XAV-939 and LiCl on differentiation of HL-60 cells induced by NSC67657 suggests that Wnt signalling pathway plays a key role in monocyte differentiction of HL-60 cells induced by NSC67657.
出处
《中国实验血液学杂志》
CAS
CSCD
北大核心
2016年第2期341-346,共6页
Journal of Experimental Hematology