重组腺病毒AdE-SH2-Caspase8-HA-GFP对耐伊马替尼的K562/G01细胞增殖的影响

The Effect of Recombinant Adenovirus AdE-SH2-Caspase8-HA-GFP on the Proliferation of Imatinib-resistant K562/G01 Cell Line

研究表达融合蛋白SH2-Caspase8的重组腺病毒AdE-SH2-Caspase8-HA-GFP对耐伊马替尼的BCR/ABL阳性的慢性粒细胞白血病(CML)K562/G01细胞增殖的影响。荧光显微镜观察病毒感染效率,Western blot检测融合蛋白的表达情况,MTT和细胞计数检测细胞的生长情况,流式细胞仪分析细胞增殖周期,甲基纤维素克隆形成实验检测细胞的克隆形成能力。结果显示,荧光显微镜观察病毒感染效率高,Western blot能检测到目的蛋白的表达,MTT和细胞计数检测可见,与对照组相比AdE-SH2-Caspase8-HA-GFP能显著抑制细胞的生长;流式周期检测发现,AdE-SH2-Caspase8-HA-GFP组G1期细胞比例增加,S期和G2期细胞比例减少;克隆形成实验可见,AdE-SH2-Caspase8-HA-GFP能显著抑制细胞克隆的形成。综上所述,重组腺病毒AdE-SH2-Caspase8-HA-GFP表达的SH2-Caspase8融合蛋白能明显抑制耐伊马替尼的K562/G01细胞的增殖。

We did this research to study the effect of SH2-Caspase8 fusion protein expressed by recombinant adenovirus AdE-SH2-Caspase8-HA-GFP on the proliferation of K562/G01 cell line, which is a BCR/ABL positive chronic myeloid leukemia cell line and resistant to imatinib. Infection efficiency was observed under fluorescent microscopy. The expression of fusion protein was analyzed by Western blot. Cell growth was detected by MTT test and cell counts. Cell cycles were determined by flow cytometry. The ability of cell colony was assessed by colony-forming assay. As a result, the infection efficiency of AdE-SH2-CaspaseS- HA-GFP on K562/G01 cells was high as confirmed by fluorescent microscopy. SH2-Caspase8 fusion protein was expressed correctly in K562/G01 cells. As compared to control groups, AdE-SH2-Caspase8-HA-GFP adenovirus significantly inhibited cell growth. The result of FCM showed that cells in G1 phase increased, while cells in S phase and G2 phase decreased. AdE-SH2-Caspase8-HA-GFP adenovirus inhibited the colony-forming ability ofK562/G01 cells obviously. Together all, AdE-SH2-Caspase8-HA-GFP which expresses SH2-Caspase8 fusion protein can significantly inhibit the proliferation of K562/G01 cells.