氧化砷和维甲酸对PLZF-RARa阳性U937细胞的作用

Effects of arsenic trioxide and ATRA on PLZF-RARα-positive U937 leukemic cells

目的:研究氧化砷(As2O3)和全反式维甲酸(AT-RA)对PLZF-RARα阳性细胞的作用。方法:稳定转染PLZF-RARα基因的U937细胞(U937/PLZF)经As2O3、ATRA处理后,Wright-Giemsa染色观察细胞形态,MTT法检测细胞生长增殖状态,流式细胞术(FCM)检测细胞周期和细胞表面分化抗原CD11b、CD64、CD14等的表达变化,荧光免疫细胞化学染色检测融合蛋白表达,细胞化学染色和硝基蓝四氮唑(NBT)还原试验观察细胞功能分化情况。结果:U937/PLZF细胞在去除四环素条件培养后,PLZF-RARα表达明显增加。As2O3(0.5μmol/L)联合ATRA(1μmol/L)使U937/PLZF细胞核质比缩小,核仁减少但未消失;生长增殖受抑;S期细胞减少;CD11b表达增高;PLZF-RARα蛋白表达减弱,分布以胞核弥漫细小颗粒为主。细胞化学染色和NBT反应变化不明显。结论:0.5μmol/L As2O3联合1μmol/L ATRA可使PLZF-RARα阳性U937细胞产生轻微的形态学分化趋势,尚不足以引起其发生功能分化。

AIM： To investigate effects of arsenic trioxide （As203 ） and all-trans retinoic acid （ATRA） on PLZF- RARcc-positive cells. METHODS, PLZF-RARcc-positive U937 cells （U937/PLZF） were used as an in vitro model. The change of cell morphology was observed by Wright-Giemsa staining. Cell growth and proliferation were detected by methyl thiazolyl tetrazolium（MTT） assay. Cell cycle distribution and expression of cell membrane surface differentiation-related antigens （such as CD11b, CD64 and CD14） were determined by flow cytometry assay. Expression of PLZF was analyzed by immunofluorescence. Functional differentiation was reflected by nitroblue tetrazolium（NBT） reduction ability and cytochemistry staining. RESULTS： While U937/PLZF cells were incubated in tetracycline-withdrawn medium, the expression of PLZF-RARα protein increased. After treated with As203（0.5 iamol/L） and ATRA （1 iamol/L）, U937/PLZF cells presented some changes such as decreased nuclear/cytoplasm ratio, and partial disappearance of nucleoli, suggesting a certain degree of morphological differentiation. The cell growth and proliferation were inhibited in a dose- and time-dependent manner. The proportion of cells in S phage was decreased and CD11 b level was increased. The expression of PLZF relocated in treated cells. However, no significant difference in NBT assay and cytochemistry staining was documented with the combination therapy. CONCLUSION： The combination of As2O3 with ATRA can cause a slight tendency to morphological differentiation but is insufficient to induce functional differentiation of PLZF-RARα positive U937 leukemia cells.