【摘要】 目的研究应用载体表达短发夹 RNA(short hairpin RNA,shRNA)干扰技术抑制人 T 淋巴细胞白血病细胞株 Jurkat 细胞 survivin 基因的表达,探讨 survivin 基因表达缄默对 Jurkat 细胞凋亡和增殖的影响。方法构建针对 survivin 基因的 shRNA 重组质粒并转染至 Jurkat 细胞,分别用多重 PCR和 Western blot 法检测瞬时转染和稳定转染细胞 survivin 基因 mRNA 和蛋白表达水平的变化;流式细胞术检测瞬时转染和稳定转染细胞凋亡指数的变化,绘制细胞生长曲线,探讨 survivin shRNA 转染对细胞生长和增殖的影响。结果多重 PCR 结果示与无功能(对照组)shRNA 处理组和磷酸盐缓冲液处理组比较,survivin shRNA 瞬时转染和稳定转染细胞 surivivin 基因 mRNA 表达均显著下降,抑制率分别为66.675% 和60.69%(P<0.05);Western blot 结果显示 survivin shRNA 瞬时转染和稳定转染细胞survivin 蛋白表达水平亦显著降低,抑制率分别为63.41% 和60.18%(P<0.05)。流式细胞术检测瞬时转染和稳定转染细胞的凋亡率显著增加,分别为(22.41±2.83)% 和(20.73±2.56)%(与对照组比较,P均<0.05),细胞倍增时间显著延长。生长曲线显示稳定转染细胞的生长显著减慢。结论shRNA 重组质粒介导的 RNA 干扰能明显抑制 Jurkat 细胞 survivin 基因 mRNA 和蛋白产物的表达,诱导细胞凋亡和生长抑制。更多还原

【Abstract】 Objective To transfect a short hairpin RNA(shRNA)against survivin gene into human T lymphoblastic leukemia cell line Jurkat,and to explore the effects on apoptosis and proliferation of transfect- ed cells.Methods The survivin-shRNA expression vector were constructed and transfected into Jurkat cells.Expression of survivin mRNA and protein were assessed by RT-PCR and Western blot analysis respec- tively.Apoptosis index of transfected Jurkat cells was quantified by flow cytometry.The potential of cell pro- liferation was described by cell growth curves.Results In survivin-shRNA transfected Jurkat cells,survivin mRNA levels were significantly reduced by 66.67%(transient transfection)and 60.69%(stable transfec- tion)respectively,compared with that in control-shRNA treated group and PBS treated group(P<0.05); and the levels of survivin protein were significantly reduced by 63.41%(transient transfection)and 60.18% (stable transfection),compared with that in the two control groups(P<0.05).Apoptosis index was signifi- cantly increased during both transient and stable transfection,respectively[(22.41±2.83)% and (20.73±2.56)%(P<0.05)].Survivin-shRNA also inhibited the proliferation of Jurkat cells.Conclu- sions Vector-based survivin-shRNA can effectively reduce the expression of survivin gene,induce apoptosis and inhibit growth of transfected Jurkat cells.更多还原