AML1-ETO真核表达载体的构建及对U937细胞增殖和分化的影响

Construction of AML1-ETO eukaryotic expression vector and its effects on proliferation and differentiation of U937 cells

目的构建pcDNA3．1-AML1-ETO真核表达载体，并观察AMLI-ETO融合蛋白在U937细胞内的表达并检测其对细胞增殖与分化的影响。方法以原核表达载体pCMV5-AMLI—ETO为模板扩增AML1-ETO目的片段，将该基因重组于pcDNA3．1／V5-His-TOPO真核表达载体上，用脂质体转染技术将其导入U937细胞，经G418筛选获得稳定转染的克隆，PCR检测AML1-ETO基因的整合，RT—PCR及Westernblot检测AML1-ETOmRNA和蛋白的表达，用锥虫蓝拒染人工计数法观察细胞增殖活性，流式细胞术检测髓系分化抗原表达的变化，瑞特染色法观察细胞形态改变。结果pcDNA3．1-AML1-ETO经酶切鉴定及DNA测序证实序列完全正确，筛选出AMLI-ETO基因高表达的亚克隆，证实AML1-ETO基因稳定转染到U937细胞中并得到表达；转染细胞增殖受抑（P〈0．05），髓系分化抗原CD11b表达阳性率[（4．17±0．31）％]低于转染空载体和未转染对照组[（11．40±0．17）％、（11．03±0．15）％]（P〈0．001），形态呈低分化表现。转染细胞经TPA处理后，CD11b表达无明显变化（P〉0．05）。结论成功构建pcDNA3．1-AML1-ETO表达载体，并在真核细胞中得到了正确表达，AML1-ETO基因能抑制U937细胞的增殖与分化，为进一步研究该基因致白血病的机制奠定了基础。

Objective To construct a pcDNA3.1 -AML1-ETO expression vector and investigate its effects on proliferation and differentiation of U937 leukemic cells. Methods AML1-ETO gene was amplified by PCR from pCMV5-AMLI-ETO and inserted into eukaryotie expression plasmid pcDNA3.1/V5-His-TOPO. The recombinant plasmid was transfected into U937 cells by Lipofectamin 2000. Individual clones selected with C,418 were isolated. The integration and the expression levels of AMLI-ETO in transfectants were deter- mined by PCR, RT-PCR and Western blot analysis respectively. T17pan blue refusal staining method was used to detect the proliferation of U937 cells. Light microscope was applied to observe the moqohologic chan- ges of the cell. The expression of myeloid cell differentiation antigen was detected using flow cytometry. Re- sults The recombinant pcDNA3. 1-AML1-ETO was confirmed by enzyme digestion and sequencing. The highly expressing AMLI-ETO subclone was established. AML1-ETO was expressed in U937 cells transfeeted with peDNA3.1 -AML1-ETO. The growth of the monoelona] ceils was inhibited evidently （P 〈 0.05 ）. The expression of CD1 lb in transfected group [ （4.17 ± 0.31 ）% ] was lower than that in empty plasmid transfeeted group and non-transfeeted group [ （ 11.40± 0.17 ） % and （ 11.03 ± 0.15 ） % ] respectively （ P 〈 0. 001 ）. Transfected cells displayed morphology of less differentiation. The expression level of CDll b was unchanged in transfected cells treated with TPA （ P 〉 0.05 ）. Conclusion The eukaryotic expression vector for AMLI - ETO gene was successfully constructed and expressed in U937. AMLI-ETO inhibits the proliferation and dif- ferentiation of transfeeted cells. It provides the basis for further study of mechanisms of AMLI-ETO in leuke-mogenesis.