论文部分内容阅读
目的研究应用短发夹RNA(shRNA)技术缄默Survivin基因表达后对白血病细胞株Jurkat细胞凋亡及对化疗药物敏感性的影响。方法构建shRNA-Survivin重组质粒,电击转染至Jurkat细胞,G418筛选得到稳定表达重组质粒的细胞系,反转录(RT)-PCR检测转染细胞中Survivin mRNA表达水平变化,化疗药物长春新碱(VCR)及柔红霉素(DNR)分别作用各转染组细胞,四甲基偶氮唑蓝(MTT)比色法测定各组细胞的增殖抑制率,流式细胞术测定药物对各组细胞的凋亡效应。结果 DNA测序证实表达质粒构建成功,RT-PCR检测结果显示重组质粒能明显抑制Jurkat细胞Survivin mRNA表达,与对照组及阴性质粒转染组比较,差异均有统计学意义(Pa<0.05)。VCR及DNR作用转染细胞后,可有效抑制细胞增殖及诱导细胞凋亡,与对照组及阴性质粒转染组比较,差异均有统计学意义(Pa<0.05)。结论 Survivin基因对Jurkat细胞生长有重要作用,转染shRNA-Survivin表达质粒能有效抑制细胞Survivin mRNA表达,缄默Survivin基因表达的细胞对化疗药物的敏感性显著增高。
Objective To investigate the effect of short hairpin RNA (shRNA) silencing of Survivin gene on the apoptosis of leukemia cell line Jurkat and its sensitivity to chemotherapeutic drugs. Methods shRNA-Survivin recombinant plasmids were constructed and transfected into Jurkat cells by electroporation. The cell lines stably expressing recombinant plasmids were screened by G418. The expression of Survivin mRNA in transfected cells was detected by reverse transcription-polymerase chain reaction (RT-PCR) (VCR) and daunorubicin (DNR). The proliferation inhibition rate of each group was determined by MTT colorimetric assay. Cell apoptosis effect. Results The DNA sequencing confirmed that the expression plasmid was successfully constructed. The RT-PCR results showed that the recombinant plasmids could significantly inhibit Survivin mRNA expression in Jurkat cells compared with the control group and the negative plasmid transfected group (P <0.05). VCR and DNR transfected cells, can effectively inhibit cell proliferation and induce apoptosis, compared with the control group and negative plasmid transfection group, the difference was statistically significant (Pa <0.05). Conclusion Survivin gene plays an important role in the growth of Jurkat cells. Transfection of shRNA-Survivin expression plasmid can effectively inhibit the expression of Survivin mRNA, and the sensitivity of cells expressing silencing Survivin gene to chemotherapeutic drugs is significantly higher.