Bcl-2 shRNA对甲氨蝶呤诱导的Raji细胞凋亡及细胞增殖作用的影响

The study on effects of Bcl-2 shRNA in enhancing methotrexate-induced apoptosis and inhibiting cell proliferation in Raji cells

目的:探讨Bcl-2短发夹样RNA(short hairpin,shRNA)对甲氨蝶呤(methotrexate,MTX)诱导的人Burkit淋巴瘤Raji细胞凋亡的增强作用。方法:采用脂质体介导的转染方法将构建有Bcl-2 shRNA的质粒转染Raji细胞,然后加入MTX,采用RT-PCR法检测作用48h时细胞Bcl-2mRNA表达水平,免疫荧光法检测细胞中Bcl-2蛋白表达水平,MTT法检测经处理24、48和72h后细胞的增殖活性,Giemsa染色及FCM法检测细胞凋亡。结果:将Bcl-2 shRNA质粒转入Raji细胞后,Bcl-2 mRNA及蛋白表达水平明显降低(P<0.05),转染Bcl-2 shRNA联用MTX可明显诱导细胞凋亡,细胞增殖活性显著降低,分别与MTX组、阴性shRNA联用MTX组、Bcl-2 shRNA组及空质粒+MTX组相比差异有统计学意义(P<0.05)。结论:Bcl-2 shRNA可增强MTX对Raji细胞增殖的抑制作用,增加对细胞凋亡的诱导作用。

Objective：To study the effect of Bcl-2 short hairpin RNA （shRNA） in enhancing methotrexate （MTX）-induced apoptosis of Raji cells. Methods：Expression plasmid containing Bcl-2 shRNA was transfected into Raji cells by lipofectmine 2000 and then the transfected cells were treated with MTX. The expression levels of Bcl-2 mRNA and protein were evaluated by RT-PCR and im- munofluorescence method at 48 h of transfection. MTT assay was used to analyze cell proliferation at 24, 48 and 72 h. Apoptosis was detected by Giemsa staining and flow cytomertric cell cycle analysis. Results：After transfection with Bcl-2 shRNA, the expression levels of Bcl-2 mRNA and protein in Raji cells were significantly decreased （P〈0.05）. Bcl-2 shRNA transfection plus MTX treatment induced marked apoptosis, decreased in cell proliferation activity, and increased in apoptotie rate. The difference was significant com- pared with MTX group, negative shRNA plus MTX group, Bcl-2 shRNA group, and empty plasmid plus MTX group （P〈0.05）. Conclusion：Bcl-2 shRNA could enhance MTX-induced apoptosis and inhibition of cell proliferation in Raji cells.