摘要
目的研究4-氨基-2-三氟甲基苯基维甲酸酯(ATPR)在诱导慢性粒细胞白血病K562细胞分化过程中蛋白酪氨酸磷酸酶(Shp2)的作用。方法 ATPR作用K562细胞,CCK-8法检测细胞生长抑制率;Q-PCR和Western blot法检测Shp2表达;合成特异性荧光短链Shp2 siRNA链,抑制Shp2表达后,观察ATPR对K562细胞增殖和形态学影响。流式细胞术测定周期动力学和特异性分化指标CD235a表达,利用Western blot和Q-PCR法检测维甲酸受体RARs蛋白和mRNA的表达。结果ATPR(10-5mol/L)作用于K562细胞72 h时能最显著地抑制增殖,且Shp2的表达明显下降。靶向沉默K562细胞中Shp2基因的最佳序列为PTPN-Homo-438,siRNA与Lipo的最佳浓度比例为1∶0.05。与ATPR单独用药组比较,siRNA-Shp2+ATPR组的K562细胞增殖活性升高,G0/G1期细胞所占比例降低,S期增多,CD235a的表达下降,形态学较空白组变化不大。Shp2转染沉默后用药组维甲酸受体RARs表达与单独用药组比较有差异。结论 Shp2沉默后,ATPR诱导K562细胞分化作用减弱,提示ATPR诱导K562细胞分化过程中,Shp2可能发挥着作用。
Objective To investigate the effect of protein tyrosine phosphatase Shp2 on 4-amino-2-trifluoromethyl- phenyl retinate （ATPR） induced differentiation of K562 cells. Methods K562 cells were cultured and treated with ATPR at different concentrations. The proliferation of K562 cells was evaluated using CCK-8. The mRNA and protein expressions of Shp2 were detected by Q-PCR and Western blot . Lipofectamine 2000 transfection reagent was used to transfect the small interfering RNA （siRNA） in K562 cells. Using CCK-8 assay to detect the proliferation of K562 cells. Morphologic changes were observed via Wright-Giemsa staining. Using FCS to observe the expression of an exclusive cell surface antigen CD235a and the distribution of cell cycle on K562 cells. The mRNA and protein expressions of retinoic acid receptors （RARα and RARγ） were detected by Q-PCR and Western blot, respectively. Results The best inhibitory effect reached the peak at 72 h. The expressions of Shp2 remarkably decreased. De- tected through fluorescence microscope and Western blot, the PTPN-Homo-438 and the concentration ratio of the siRNA and Lipo was 1 ： 0.05. The gene silencing efficacy was the best. Compared with the ATPR group, the pro- liferation of K562 cells were not inhibited by treatment with ATPR （ 10 -5 moL/L） . The percentage of cells in CA）/ G1 phase was decreased while S-phase cells were increased, and the expression level of the maturation specific cell surface marker CD235a decreased in K562 cells. Q-PCR and Western blot results showed that RARα mRNA and protein expressions of siRNA ＋ ATPR group were significantly increased than ATPR group. RARe/mRNA and pro- tein expressions of siRNA ＋ ATPR group were decreased than ATPR group. Conclusion The effect of ATPR in- duced differentiation of K562 cells might be involved with the Shp2 gene.
作者
丁然
王静静
葛金芳
陈飞虎
Ding Ran Wang Jingjing Ge Jinfang et al(School of Pharmacy,Anhui Medical University, Hefei 23003)
出处
《安徽医科大学学报》
CAS
北大核心
2016年第10期1403-1410,共8页
Acta Universitatis Medicinalis Anhui
基金
国家科技部"重大新药创新"科技重大专项(编号:2011ZX09401-021)
