WT1异构体比例转变对人白血病细胞株HL-60增殖和凋亡的影响

 目的： 探讨WT1异构体比例改变对人白血病细胞株HL-60增殖和凋亡的影响。方法： 逆转录获得人WT1(17AA－/KTS－)异构体全长cDNA序列,连接到PCDH1-MCS1-EF1-copGFP慢病毒质粒载体上, 酶切和测序证实正确构建重组质粒，建立WT1异构体表达比例改变的单克隆细胞株。MTT法检测细胞增殖活性，形态学观察及Annexin V 检测细胞凋亡。结果： 成功构建了真核表达载体PCDH1-MCS1-EF1-copGFP-WT1(17AA－/KTS－)，转染HL-60细胞后，实时荧光定量RT-PCR和Western blotting 显示重组质粒转染细胞中WT1(17AA－/KTS－) mRNA和蛋白水平明显高于空质粒转染组和正常对照组。重组质粒转染细胞的生长抑制率高于空质粒转染组和正常对照组, 差异显著(P<0.05)。加2 μmol/L 三氧化二砷（As2O3）培养48 h后, 形态学观察发现3组细胞都出现了凋亡形态, 但重组质粒转染细胞更为明显。流式细胞术结果显示，As2O3作用24 h后，重组质粒转染细胞凋亡率较空质粒转染组和正常对照组细胞升高，差异显著(P<0.05)。结论： 增加外源性WT1(17AA－/KTS－)异构体的表达能抑制HL-60细胞增殖，促进As2O3 诱导的细胞凋亡。

The effect of WT1 gene isoform transfection on proliferation and apoptosis of leukemia cell line HL-60

Abstract] AIM: In order to study the potential effects of exogenous WT1 gene isoforms on proliferation and apoptosis in leukemia cell line HL60. METHORDS: WT1(-17AA/-KTS) gene obtained by RT-PCR was cloned into PCDH1-MCS1-EF1-copGFP plasmid. The recombinant plasmid was confirmed by enzyme digestion and sequencing，and then it was transfected into HL-60 cells by Lipofectamine TM2000. The stable transformants were selected by G418 screening. WT1 (-17AA/-KTS) expression was identified by real time fluorescence quantitative PCR and Western blotting analysis. The proliferating ability was measured by MTT assay. The apoptosis of HL-60 cells was observed by morphology and the apoptotic percentage was determined by flow cytometry. RESULTS: The eukaryotic expression vector PCDH1-MCS1-EF1-copGFP-WT1 (-17AA/-KTS) was successfully constructed. Real time fluorescence quantitative PCR and Western blotting analysis showed that recombinant cells exhibited high mRNA and protein levels of WT1(-17AA/-KTS). The growth of recombinant cells was slower than that of HL-60 cells trasfected with vacant vector and normal HL-60 cells. After exposure to As2O3 at 2μmol/L for 48 hours, the recombinant cells and control cells both exhibited the morphological hallmarks of apoptosis, but the former was more typical than the latter. Flow cytometry analysis showed the recombinant cells enhanced the apoptosis rate after exposure to As2O3 for 24 hours than control cells. CONCLUSION: Exogenous WT1 (-/-) gene could inhibit the proliferation and promote the apoptosis of leukemia cells.