WT1基因异常表达在阵发性睡眠性血红蛋白尿症发病机制中的作用

Abnormal WT1 gene expression in paroxysmal nocturnal hemoglobinuria

目的 探讨WT1基因异常表达在阵发性睡眠性血红蛋白尿症（PNH）发病机制中的作用.方法 采用RT-PCR法检测CD59及CD59-骨髓单个核细胞（BMMNC）WT1 mRNA的表达水平;利用RNA干扰（RNAi）技术敲低WT1基因,采用流式细胞术检测WT1基因敲低前后BMMNC细胞周期及凋亡率.结果 PNH患者CD59细胞组、CD59＋细胞组及正常对照组WT1 mRNA的相对表达量分别为1.06±0.12、0.90±0.12和0.86±0.05,CD59细胞组明显高于CD59＋细胞组和正常对照组（P值均＜0.05）,而CD59＋细胞组和正常对照组比较差异无统计学意义（P＞0.05）.PNH患者WT1 mRNA的相对表达量与CD59细胞数量呈正相关（r2=0.490,P=0.016）,而与CD59＋细胞数量无明显相关性.PNH患者骨髓CD59细胞转染靶向WT1基因小干扰RNA（siRNA）后,WT1 mRNA表达水平下降;PNH患者空白对照组和转染无义序列组（siRNA-scr转染组）G0/G1期细胞比例分别为（92.73±3.71）％和（93.06±4.14）％,S期分别为（6.99±3.61）％和（6.73±4.08）％;siRNA-WT1转染组G0/G1期细胞比例为（94.46±3.71）％,S期为（5.40±3.55）％,与空白对照组和siRNA-scr转染组比较差异均有统计学意义（P＜0.05）.siRNA-WT1转染组细胞凋亡率[（35.91±22.36）％]较空白对照组[（27.39±18.99）％]和siRNA-scr转染组[（26.12±17.10）％]明显增加（P＜0.05）.结论 转染WT1 siRNA能有效抑制PNH患者CD59-细胞中WT1基因的表达,减弱其增殖能力,促进其凋亡.WT1基因过表达可能参与PNH克隆的增殖过程.

Objective To explore the pathogenesis of abnormal WT1 expression in paroxysmal nocturnal hemoglobinuria （PNH）.Methods The expression of WT1 mRNA in CD59-and CD59＋ bone marrow mononuclear cells （BMMNC） were measured by semi-quantitative reverse transcription PCR.After WT1 gene silence by RNA interference （RNAi） technology,biological characteristics of BMMNC were investigated by flow cytometry.Results The relative expression of WT1 mRNA in PNH CD59 BMMNC （1.06±0.12） was significantly higher than that in PNH CD59＋ BMMNC （0.90±0.12） and normal BMMNC （0.86±0.05,P＜0.05）,but there was no significant difference between PNH CD59＋ BMMNC and normal BMMNC （P＞0.05）.WT1 mRNA expression in PNH was positively correlated with the proportion of CD59-cells （r2=0.490,P=0.016）,but had no relationship with the proportion of CD59＋cells.After WT 1 gene silence by siRNA in PNH CD59 BMMNC,WT1 mRNA expression was decreased.The proportions of G0/G1 phase in PNH CD59-cell blank control group and siRNA-scr transfected group were （92.73±3.71） ％ and （93.06±4.14） ％,and the proportions of S phase were （6.99±3.61）％ and （6.73±4.08）％,respectively.The proportions of G0/G1 and S phase in siRNA-WT 1 transfected group was （94.46±3.71）％ and （5.40±3.55）％,respectively.There were significant differences in the proportions of G0/G1 phase and S phase among the controls,siRNA-WT1 transfected group and siRNA-scr transfected group （P＜0.05）.The rate of apoptosis in siRNA-WT1 transfected group [（35.91±22.36）％] was significantly higher than those in controls [（26.12 ± 17.10）％] and siRNA-scr transfected group [（27.39 ± 18.99）％] （P＜0.05）.Conclusions siRNA-WT1 could effectively suppress the WT1 gene expression of CD59 clone in PNH patients,inhibit its proliferation,and promote its apoptosis.WT1 gene expression might contribute to PNH clone proliferation.