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目的 探讨反义hTERT基因体外对白血病细胞端粒酶基因表达及其活性的抑制作用。方法 采用基因重组技术设计并构建了针对端粒酶活性蛋白催化亚单位mRNA的 5′端序列的反义pcDNA hTERT真核表达载体 ,通过SuperFect(QIAGEN)将其瞬时转染人早幼粒白血病细胞株HL60 及人红白血病细胞株K562 ,并运用流式细胞术测定其细胞凋亡与细胞周期的变化 ,同时运用实时荧光定量逆转录 聚合酶链反应方法对其端粒酶蛋白催化亚单位基因的表达水平进行了检测 ,端粒酶活性的检测则采用了非放射性TRAP 银染法。结果 成功地构建了反义pcDNA hTERT真核表达载体 ,并将其转染至K562 和HL60 白血病细胞株。结果与空载体组、空白组相比 ,反义 pcDNA hTERT组体外显示出明显有效地抑制端粒酶的基因表达及其活性的作用 ;在细胞凋亡与细胞周期方面 ,反义组能引起细胞周期左移 ,增殖分裂能力降低 ,同时伴有细胞凋亡数的增加。结论 研究构建的反义hTERT基因体外确实能明显有效地下调白血病细胞端粒酶的基因表达及其活性 ,在抗肿瘤基因治疗中具有特异的靶向性和潜在应用的广谱性
Objective To investigate the inhibitory effect of antisense hTERT gene on telomerase gene expression and activity in leukemia cells in vitro. Methods The antisense pcDNA hTERT eukaryotic expression vector targeting the 5 ’end of telomerase catalytic subunit mRNA was designed and constructed by gene recombination technique and transiently transfected into human promyelocytic leukemia cells by SuperFect (QIAGEN) Strain HL60 and human erythroleukemia cell line K562, and the changes of apoptosis and cell cycle were detected by flow cytometry. Meanwhile, the expression of telomerase catalytic subunit gene was detected by real-time fluorescence quantitative reverse transcription polymerase chain reaction The expression level was tested. The telomerase activity was detected by non-radioactive TRAP silver staining. Results The antisense pcDNA hTERT eukaryotic expression vector was successfully constructed and transfected into K562 and HL60 leukemia cell lines. Results Compared with the empty vector group and the blank group, the antisense pcDNA hTERT group showed a significant inhibitory effect on the gene expression and activity of telomerase in vitro. In the apoptosis and cell cycle, the antisense group can cause the cells Left cycle, reduced proliferation and division, accompanied by an increase in the number of apoptotic cells. Conclusion The constructed antisense hTERT gene can effectively and efficiently down-regulate the gene expression and activity of telomerase in leukemia cells in vitro and has specific targeting and potential broad-spectrum applications in anti-tumor gene therapy