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目的研究反义hTERT基因对白血病细胞体外增殖的抑制作用。方法体外通过SuperFect将已构建好的正、反义hTERT真核表达载体转染HL60白血病细胞,再经过G418及PCR筛选鉴定分别转入了正、反义hTERT载体的抗性克隆细胞HL60-s和HL60-as。随后运用实时荧光定量RT-PCR技术及TRAP-银染法对各组细胞内源性hTERT mRNA的表达情况及端粒酶的活性进行检测。同时还采用MTT法、双层软琼脂克隆形成试验、流式细胞术观察和分析反义hTERT对白血病细胞体外生长增殖活力的影响及是否能诱导瘤细胞的凋亡。结果与空白对照、正义hTERT组相比,反义hTERT能显著地降低HL60细胞内源性hTERT mRNA的表达(P<0.01)和下调端粒酶活性。当各组细胞传至第25代后,与HL60、HL60-s比较,HL60-as细胞的生长速度和集落形成能力明显地减慢和降低,同时伴有凋亡率的显著增加。结论反义hTERT在体外能抑制白血病细胞的生长增殖能力,其潜在、广谱的抗肿瘤作用的分子生物学机制可能是首先通过抑制和下调hTERT表达(端粒酶活性)而最终引发瘤细胞衰亡的途径来实现的。
Objective To study the inhibitory effect of antisense hTERT gene on the proliferation of leukemia cells in vitro. Methods The positive and antisense hTERT eukaryotic expression vector was transfected into HL60 leukemia cells by SuperFect in vitro, then identified by G418 and PCR screening, and then transfected into positive and antisense hTERT vector-resistant clones HL60-s and HL60-as. Subsequently, the expression of endogenous hTERT mRNA and the activity of telomerase in each group were detected by real-time fluorescence quantitative RT-PCR and TRAP-silver staining. At the same time, we also used MTT method, double soft agar colony formation assay, observed and analyzed by flow cytometry antisense hTERT on leukemic cells in vitro proliferation and proliferation of vitality and whether can induce tumor cell apoptosis. RESULTS: Antisense hTERT significantly decreased the expression of endogenous hTERT mRNA (P <0.01) and down-regulated telomerase activity in HL60 cells compared with the blank control group and the normal hTERT group. Compared with HL60 and HL60-s, the growth and colony-forming ability of HL60-as cells obviously slowed down and decreased, accompanied by the significant increase of apoptosis rate. CONCLUSION: Antisense hTERT can inhibit the proliferation and proliferation of leukemia cells in vitro. The molecular mechanism of its potential and broad-spectrum antitumor activity may be that the tumor cells will eventually be induced to decline by inhibiting and down-regulating hTERT expression (telomerase activity) Way to achieve.