摘要目的 探讨靶向干扰IER3IP1基因对K562细胞增殖和红系分化的影响.方法 针对IER3IP1基因设计并构建小发夹状(shRNA)真核表达载体,转染K562细胞后用RT-PCR方法鉴定沉默效果.采用MTT实验、联苯胺染色、光镜和电镜观察、流式细胞术以及RT-PCR,观察抑制IER3IP1基因表达后对K562细胞的影响;采用0.2μmoL/L伊屿替尼诱导K562细胞2 d,观察诱导分化过程中IER3IP1基因表达变化情况.抑制IER3IP1基因表达后对红系分化相关基因Gfi-1B、GPA和γ-globin以及细胞表面GPA蛋白表达的影响.结果 成功构建针对IER3IP1基因的shRNA真核表达载体,转染至K562细胞中,该基因mRNA表达水平明显降低,抑制率达76%(P<0.01).与对照组细胞相比,K562-shRNA-IER3IP1组bcr-abl mRNA表达升高(P<0.05);MTT检测结果显示细胞增殖能力增强(P<0.01);流式细胞术检测结果显示S期细胞比例增加,G0/G1期细胞比例减少(P<0.05);电镜可见细胞常染色质增加、异染色质减少,内质网部分扩张,囊泡样结构滞留.0.2μmol/L伊马替尼作用48 h后,K562-shRNA-IER3IP1组联苯胺染色阳性率由作用前的(44.7±2.5)%降低至(22.7±1.5)%(P<0.01),且红系分化相关基因Gfi-1B、GPA和γ-globin的mRNA表达水平明显降低,细胞表面GPA蛋白表达水平降低.同时伊马替尼作用过程中IER3IP1基因在作用3 h时表达明显升高,6 h时降低,12～48 h表达水平基本不变.结论 干扰IER3IP1基因表达后,K562细胞增殖加快,红系分化过程受阻,提示该基因可能在K562细胞增殖和向红系分化过程中发挥作用.

abstractsObjectives To investgate the effect of IER3IIP1 gene silence by RNA inferference on erythroid differentiation and proliferation of K562 cells. Methods The shRNA eukaryotic expression vectors targeting IER3I0P1 gene were constructed. Silence effect was detected by semiquantitative RT-PCR after the vectors being transfected into K562 cells. The impact on K562 cells was studied by MTT assay, benzidine staining,light microscope and electron microscopy observation, flow cytometry and RT-PCR, respectively. The expression of erythroid differentiation-related genes Gfi-1B,GPA and γ-globin and GPA protein expressed on these cells were studied after being exposed to 0.2 μmol/L imatinib for 48 hours. Results The expres-sion of IER3IP1 gene was significantly inhibited with a inhibition rate of 76% (P<0.01). Compared with the control group, bcr-abl mRNA level was significantly increased in K562-shRNA-IER3IP1 group(P<0.05). The proliferation capacity was significantly enhanced(P<0.01)and the proportion of cells at G0/G1 phase sig-nificandy decreased while at S phase significantly increased (P<0.05) in K562-shRNA-IER3IP1 group. Un-der the electron microscopy observation, the number of euchromatin was increased while heterochromatin de-creased. There were structural abnomalities in endocytoplasmic reticulum and clusters of vesicular. The pro-portion of benzidine staining positive cells decreased from (44.7±2.5) % in control group to (22.7±1.5 )% in tested group (P<0.01). mRNA expression of Gfi-1B ,GPA and γ-globin gene and the expression of GPA protein on cell surface were all significantly decraesed after being exposed to 0.2 μmol/L imatinib for 48 hours in K562-shRNA-IER31PI group (P<0.01). During the erythroid differentiation induced by imatinib,mRNA expression of IER3IP1 obviously increased at the 3 h, and decreased from 6 h to 48 h. Con-clusions Inhibition of expression of IER3IP1 gene can inhibit erythroid differentiation and elevate the prolif-eration of K562 cells. The IER3IP1 gene may play a role on erythroid differentiation and proliferation of K562 cells.