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

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48 h时细胞 Bcl-2 mRNA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72 h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.

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

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 immunofluorescence 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 apoptotic rate. The difference was significant compared 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.