曲古菌素A和ATRA合用对PLZF-RARα转基因阳性发病鼠骨髓细胞的诱导分化作用

Differentiation induced by the combination of trichostatin A with all-trans retinoic acid in leukemia cells of PLZF-RARα transgenic mice

目的:研究组蛋白去乙酰化酶抑制剂曲古菌素A(trichostatin A,TSA)和ATRA对PLZF-RARα转基因发病鼠骨髓细胞的作用.方法:分子克隆技术构建hCG-PLZF-RARα转基因构件.显微注射建立仅在髓系细胞中表达PLZF-RARα的转基因小鼠模型.DNA-PCR,Southern blot,RT-PCR,免疫荧光,血象,骨髓象,脾细胞形态,病理等检测分析基因整合表达及发病情况.取PLZF-RARα转基因发病鼠骨髓细胞体外培养,经TSA和ATRA作用一段时间后,Wright-Giemsa染色观察细胞形态,流式细胞仪检测细胞周期、细胞表面分化抗原CD11b和CD117等的表达,荧光免疫细胞化学染色检测融合蛋白表达,硝基蓝四氮唑(NBT)还原试验观察细胞功能分化情况.结果:TSA(30μg/L)联合ATRA(1μmol/L)使发病鼠骨髓细胞核质比缩小,幼稚细胞比例减少,细胞生长增殖受抑,S期细胞减少,PLZF蛋白的表达呈局部聚集的趋势,但NBF还原试验变化尚不明显.结论:组蛋白去乙酰化酶抑制剂TSA联合ATRA可使PLZF-RARα转基因发病鼠骨髓细胞发生部分分化.

AIM： To investigate the effect of all-trans retinoic acid （ATRA） in combinetion with trichostatin A （TSA）, a histone deacetylase inhibitor, on PLZF-RARα-positive cells. METHODS： Molecular cloning technology was used to construct hCG-PLZF-RARα gene. HCG-PLZF-RARα transgenic mice were generated, in which PLZF-RARα fusion gene was driven by hCG promoter to express in myeloid cells of mice. The genotype and phenotype were analyzed by PCR, Southern Blot, RT-PCR, im- munofluorescence, morphology of bone marrow ceils, peripheral blood cells and spleen ceils, pathology of bone, spleen and liver. Bone marrow leukemia cells were cultured in vitro. The change of cell morphology was observed by Wright-Giemsa staining; cell cycle distribution and expression of cell membrane surface differenti- ation-related antigens （such as CD11b, CD117 and Gr-1 ） were determined by flow cytometry assay. Expression of PLZF was analyzed by immunofluorescence. Functional differentiation was reflected by nitroblue tetrazolium （NBT） reduction ability. RESULTS：Some hCG- PLZF-RARα transgenic mice developed leukemia resembling human chronic myeloid leukemia. After treated with TSA and RA, bone marrow leukemia cells presented morphologically some features of differentiated ceils; the cell growth and proliferation were inhibited in a time-dependent manner. The proportion of cells in S phage was decreased. The expression of PLZF relocated in treated ceils. However, no significant difference in NBT assay was documented with the combination therapy. CONCLUSION： Combination of histone deacetylase inhibitor with ATRA can cause a partial differentiation of PLZF-RARα positive ceils.