全反式维甲酸诱导HL60细胞分化分子机制探讨

Mechanism of HL60 cells differentiation induced by ATRA

目的探讨全反式维甲酸(alltrans retinoic acid,ATRA)诱导HL-60细胞分化过程中PADI4的变化与作用机制。方法 ATRA诱导HL-60细胞24、48、72和96h后,采用Wright-Gimesa染色观察细胞形态学变化;采用流式细胞术检测细胞表面分化抗原CD11b的表达变化;采用RT-PCR分析PADI4在基因水平的表达趋势,蛋白质印迹法检测PADI4在蛋白水平的表达变化;RNAi技术干扰PADI4后流式细胞仪检测CD11b变化,蛋白质印迹法检测PADI4、p42/44、p65、p105和p55等蛋白水平变化。结果 HL-60细胞经ATRA诱导后与对照组相比,Wright-Gimesa染色显示核质比明显减小,核有凹陷及分叶,杆状核、分叶核现象明显增多。流式细胞术检测结果显示,细胞表面分子CD11b表达由2.1%升高到48.9%,升高了346.8%,t=411.51,P<0.01;RT-PCR结果显示,PADI4在mRNA水平表达随时间延长逐渐升高,F=318.046,P<0.001,r=0.595;PADI4与内参基因GAPDH灰度值的比值从0.122±0.033升高到0.464±0.077,差异有统计学意义,F=16.002,P<0.001。蛋白质印迹法检测结果显示,PADI4在蛋白水平表达随时间延长逐渐升高,F=16.002,P<0.001,r=0.873;PADI4与内参GAPDH灰度值的比值分析从0.077±0.045升高到0.263±0.095,差异有统计学意义,F=221.8,P<0.000 1。蛋白质印迹法检测结果显示,P-p44/42与内参GAPDH灰度值的比值分析从0.470±0.023下降到0.320±0.012,差异有统计学意义,F=92.942,P<0.001。结论 ATRA诱导HL-60细胞定向粒系分化的过程中,PADI4促进HL-60细胞向粒系分化,而且PADI4可能是通过促进MAPK信号通路中的p42/44磷酸化而发挥作用。

OBJECTIVE To detect the mechanism of PADI4 signaling pathways during the process of differentiation of HL60 cells induced by ATRA.METHODS After induced by ATRA for 24,48,72 and 96h,the morphology changes of HL-60 cells were observed by Wright-Giemsa staining.The cell differentiation marker CD11 bwas analyzed by flow cytometry.The mRNA and protein expression of PADI4 was detected by RT-PCR and Western Blot assay respectively.The RNAi of PADI4 was used to interfere the expression of PADI4,the protein level of PADI4,p42/44,AKT,p65,p105 and p55were detected by Western Blot and the cell surface marker CD11 bwas analyzed by flow cytometry.RESULTS After HL-60 cells induced by ATRA compared with the control group,Wright-Gimesa staining showed the ratio of nuclear to cytoplasmic was significantly reduced.Nuclear dented and leaf,rod-shaped nucleus,lobulated phenomenon increased significantly.Flow cytometry analysis results showed that the cell surface molecule CD11 bexpression increased from 2.1%to 48.9%,increased by 346.8%,t=411.51,P〈0.01.RT-PCR showed that PADI4 expressed in mRNA level with the extension of time gradually increased（F=318.046,P〈0.001,r=0.595）.The ratio of PADI4＇s gray value to reference gene GAPDH＇s was increased from 0.122±0.033 to 0.464±0.077.The difference was statistically significant（F=16.002,P〈0.001）.Western blotting showed that PADI4 expression at the protein level increased with the extension of time gradually increased（F=16.002,P〈0.001,r=0.873）.The ratio of PADI4＇s gray value to reference gene GAPDH＇s was increased from 0.077±0.045 to 0.263±0.095.The difference was statistically significant（F=221.8,P0.000 1）.Western blotting test results showed that the ratio of P-p44/42＇s gray value to GAPDH＇s was decreased from 0.470±0.023 to 0.320±0.012.The difference was statistically significant（F=92.942,P〈0.001）.CONCLUSION During the differentiation of HL-60 cells induced by ATRA,PADI4 may promote the differentiation of HL-60 cells by increasing p42/44 phosphorylation in MAPK signaling pathway.