摘要
目的:探讨人参皂苷Rg1诱导急性髓系白血病M6型人红白血病TF-1细胞凋亡及其对红细胞生成素受体(erythropoietin receptor,EPOR)通路的影响,以及可能的机制。方法:不同浓度人参皂苷Rg1处理TF-1细胞后,应用CCK-8(cell counting kit-8)法检测细胞的增殖活性,FCM法检测并在透射电子显微镜下观察人参皂苷Rg1诱导TF-1细胞的凋亡,FCM法和免疫荧光法检测TF-1细胞中EPOR蛋白的表达水平和分布情况,实时荧光定量-PCR法检测TF-1细胞EPOR mRNA的表达,蛋白质印迹法检测人参皂苷Rg1作用后TF-1细胞的红细胞生成素(erythropoietin,EPO)反应性以及TF-1细胞中EPOR、磷酸化EPOR、酪氨酸激酶JAK2(Janus tyrosine kinase 2)、磷酸化JAK2、信号转导和转录激活因子5(signal transducers and activators of transcription 5,STAT5)、磷酸化STAT5、Bax、Bcl-2和caspase-3(激活型)蛋白的表达。结果:人参皂苷Rg1(12.5、25、50、100和200μmol/L)作用24、48和72 h后,TF-1细胞的增殖受到抑制(P<0.05)。人参皂苷Rg1(12.5、25和50μmol/L)作用48 h后,TF-1细胞的早期凋亡率高于人参皂苷Rg1未处理的对照组(P<0.05),透射电子显微镜下可见TF-1细胞出现凋亡变化。FCM法和免疫荧光检测结果均显示,人参皂苷Rg1作用后TF-1细胞膜表面EPOR的表达明显减少,并且TF-1细胞EPOR mRNA的表达水平下调(P<0.05)。与对照组比较,人参皂苷Rg1作用后TF-1细胞EPOR总蛋白的表达水平无明显变化,但降低了TF-1细胞对EPO的反应性,磷酸化EPOR的表达水平明显下调,Bax和caspase-3(激活型)蛋白的表达水平明显上调,Bcl-2以及EPOR下游JAK2、磷酸化JAK2、STAT5和磷酸化STAT5的表达水平均明显下调(P均<0.05)。结论:人参皂苷Rg1可明显抑制TF-1细胞的增殖并促进其凋亡,其促凋亡机制可能与降低TF-1细胞对EPO的反应性,下调EPOR下游相关蛋白的表达并激活caspase-3的表达有关。
Objective： To investigate the effects of ginsenoside Rgl on the apoptosis of acute myeloid leukemia M6-type human erythroleukemia TF-1 cells and the erythropoietin receptor （EPOR） pathway and to explore the possible mechanisms. Methods： After TF-1 cells treated with different concentrations of ginsenoside Rgl, the proliferation activity of TF-1 cells was detected by cell counting kit-8 （CCK-8） assay. The apoptotic change of TF-1 cells after treatment with ginsenoside Rgl was demonstrated by flow cytometry （FCM） and observed under a transmission electron microscope. The expression level of EPOR on TF-1 cell membrane after treatment with ginsenoside Rgl was detected by FCM and immunofluoresence staining. The expression levels of EPOR mRNA and EPOR, phospho-EPOR （p-EPOR）,Janus tyrosine kinase 2 （JAK2）, phospho-JAK2 （p-JAK2）, signal transducers and activators of transcription 5 （STAT5）, phospho-STAT5 （p-STAT5）, Bcl-2, Bax and cleaved caspase-3 proteins and the reactivity to erythropoietin （EPO） in TF-1 ce^ls after treatment with ginsenoside Rgl were measured by real-time fluorescent quantitative-PCR and Western blotting, respectively. Results： The proliferation activities of TF-1 cells after treatment with ginsenoside Rgl （12.5, 25, 50, 100 and 200 ~mol/L） for 24, 48 and 72 h were inhibited （P 〈 0.05）. The apoptosis rates of TF-1 cells after treatment with ginsenoside Rgl （12.5, 25 and 50 ~mol/L） for 48 h were higher than that of the control cells （without any treatment） （P 〈 0.05）. The apoptotic changes of TF-1 cells after treatment with ginsenoside Rgl could be found under a transmission electron microscope. As determined by FCM and immunofluoresence staining analyses, the expression level of EPOR on TF-1 cell membrane was decreased. The expression level of EPOR mRNA in TF-1 cells was decreased （P 〈 0.05）. As compared with the control cells, the expression level of total EPOR protein in TF-1 cells after treatment with ginsenoside Rgl had no change, but the reactivity to EPO was reduced and the expression level of p-EPOR was significantly decreased, the expressions of Bax and cleaved caspase-3 were significantly enhanced, and the expression levels of Bcl-2, JAK2, p-JAK2, STAT5 and p-STAT5 proteins were significantly decreased （all P 〈 0.05）. Conclusion： The ginsenoside Rgl can significantly inhibite the proliferation of TF-1 cells and promote apoptosis. This mechanism may be related to a decrease in reactivity to EPO, down-regulation of the expressions of EPOR downstream- related proteins and the activation of caspase-3.
出处
《肿瘤》
CAS
CSCD
北大核心
2014年第2期113-120,共8页
Tumor
基金
国家自然科学基金面上项目(编号:81171929)
重庆市教委科学技术研究项目(编号:KJ110328)
