Apg-2基因沉默对BaF3-MIGR1及BaF3-P210细胞增殖的影响

目的探讨Apg-2基因沉默对pMIGR1空载体感染的BaF3-MIGR1细胞及稳定表达Bcr-Abl融合基因的BaF3-P210细胞增殖的影响,为进一步研究Apg-2在Bcr-Abl阳性的CML细胞中的作用奠定基础。方法针对Apg-2基因609～629、845～865和2 110～2 130核苷酸合成3条shRNA序列Hspa41、Hspa42、Hspa43,同时合成阴性对照序列HspaHK,电穿孔转染BaF3-MIGR1和BaF3-P210细胞,经G418筛选稳定抑制细胞株,RT-PCR和Western blot检测细胞中Apg-2基因mRNA的转录水平及蛋白的表达水平;Am-blue法检测细胞增殖;甲基纤维素法检测细胞克隆形成能力;流式细胞技术检测细胞周期的变化。结果转染的3条shRNA质粒中,shRNA-Hspa42的干扰效果最佳。Apg-2表达抑制后,BaF3-P210细胞的增殖与克隆形成能力均明显下降(P<0.05),处于G1期细胞的百分率明显升高(P<0.05),处于S期的细胞百分率明显下降(P<0.05);而BaF3-MIGR1细胞的增殖与克隆形成能力均明显增强(P<0.05),处于S期的细胞百分率明显升高(P<0.05)。结论干扰Apg-2基因的表达可抑制Bcr-Abl阳性细胞BaF3-P210的增殖,而对Bcr-Abl阴性细胞BaF3-MIGR1的增殖起促进作用。

Effect of Apg-2 gene silence on proliferations of BaF3-MIGR1 and BaF3-P210 cells

Objective To investigate the effect of Apg-2 gene silence on proliferations of BaF3-MIGR1 cells transfected with empty vector pMIGR1 and BaF3-P210 cells for stable expression of Bcr-Abl fusion gene,and lay a foundation of further study on role of Apg-2 in Bcr-Abl positive CML cells.Methods Three shRNA sequences Hspa41,Hspa42 and Hspa43 targeting to nucleotides 609 ～ 629,845 ～ 865 and 2 110 ～ 2 130 of Apg-2 gene respectively,as well as negative control sequence HspaHK,were synthesized and transfected to BaF3-MIGR1 and BaF3-P210 cells by electroporation.The cell strains stably inhibiting Apg-2 gene were screened with G418,and determined for expressions of Apg-2 at mRNA and protein levels by RT-PCR and Western blot respectively,for proliferation by Am-blue method,for clone formation ability by methyl cellulose assay,and for cell cycle by flow cytometry.Results Of the three shRNA plasmids,shRNA-Hspa42 shwoed a satisfactory interfering effect.After Apg-2 expression was inhibited,both the proliferation and clone forming ability of BaF3-P210 cells decreased significantly(P < 0.05),while the percentage of cells at G1 phase increased signifi-cantly(P < 0.05) and those at S phase decreased significantly(P < 0.05).However,both the proliferation and clone for-mation ability of BaF3-MIGR1 cells increased significantly(P < 0.05),while the percentage at S phase increased signifi-cantly(P < 0.05).Conclusion The interference of Apg-2 gene expression inhibited the proliferation of Bcr-Abl positive BaF3-p210 cells while promoted the proliferation of Bcr-Abl negative BaF3-MIGR1 cells.