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目的探讨人端粒酶逆转录酶(hTERT)基因反义寡核苷酸对皮肤T细胞淋巴瘤(CTCL)细胞株端粒酶活性及细胞生长的影响,为CTCL基因治疗提供新的基因靶点。方法采用脂质体介导的基因转染方法,将不同浓度(10 μmol／L、20 μmol／L、30 μmol／L)的寡核苷酸分别导入CTCL细胞株Hut78中; 分别于不同的时间,应用端粒酶重复序列扩增及酶联免疫吸附方法(PCR-ELISA)、逆转录-聚合酶链反应 (RT-PCR)、流式细胞仪技术等动态观察转染细胞中端粒酶活性、hTERT mRNA表达以及细胞凋亡的变化。结果经hTERT反义寡核苷酸(AODN)作用72 h后,细胞端粒酶的活性明显下降,hTERT mRNA的表达减弱,并可观察到细胞凋亡,凋亡细胞发生率为13．05％。细胞生长抑制作用有明显的时效性,以 30μmol／L、72 h时下降最明显。而有义寡核苷酸(SODN)组和对照组以上指标均无明显变化(P>0．05)。结论 hTERT反义寡核苷酸可显著抑制CTCL细胞的端粒酶活性,抑制细胞生长增殖,诱导细胞凋亡。
Objective To investigate the effect of human telomerase reverse transcriptase (hTERT) gene antisense oligonucleotide on telomerase activity and cell growth in human cutaneous T cell lymphoma (CTCL) cell lines and to provide a novel gene target for CTCL gene therapy . Methods The oligonucleotides of 10 μmol / L, 20 μmol / L and 30 μmol / L were introduced into Hut78 cell line of CTCL by liposome-mediated gene transfection method. At different time , Telomerase activity was detected by RT-PCR and flow cytometry using telomerase repeat amplification and enzyme-linked immunosorbent assay (PCR-ELISA) , HTERT mRNA expression and apoptosis. Results After treated with hTERT antisense oligodeoxynucleotide (AODN) for 72 h, the activity of telomerase was significantly decreased and the expression of hTERT mRNA was decreased. The apoptosis rate was 13.05% . Cell growth inhibition obvious timeliness, 30μmol / L, 72 h when the most obvious decline. However, there was no significant change in the above indexes between the sense oligonucleotide (SODN) group and the control group (P> 0.05). Conclusion hTERT antisense oligonucleotide can significantly inhibit the telomerase activity of CTCL cells, inhibit cell proliferation and induce apoptosis.