Survivin基因的克隆及其反义核酸对人喉癌细胞系的作用

Cloning of human survivin gene and the role of survivin AS-ODN on Hep-2 cell lines

目的:克隆survivin基因并鉴定,初步探讨survivin基因反义核酸(AS-ODN)对人喉癌细胞系的生长抑制作用。方法:从培养的人喉癌Hep-2细胞中提取总RNA,经RT-PCR获得survivin基因;将该基因克隆到pGEM-T-Easy载体中,进行测序鉴定。以高表达survivin基因的人喉癌细胞系Hep-2为靶细胞、survivin基因AS-ODN为阻断剂,通过MTT试验观察AS-ODN对喉癌细胞的生长抑制作用,RT-PCR分析survivin基因AS-ODN对survivin基因表达的影响。结果:DNA测序结果证明,获得了survivin基因,其序列与GenBank中报道序列完全一致;MTT试验及RT-PCR分析表明,Hep-2在AS-ODN作用下,细胞数量明显减少,survivin基因AS-ODN明显抑制了喉癌细胞Hep-2的生长及其survivin基因的表达(P<0.05)。结论:Survivin基因的克隆获得了成功,为进一步探讨survivin基因在喉癌及其他恶性肿瘤诊断和治疗中的应用奠定了基础。Survivin基因AS-ODN对人喉癌细胞的生长可能起十分重要的抑制作用,有望成为喉癌基因治疗的新靶点。

Objective To clone and identify human survivin gene and to investigate the Inhibitory effect of survivin AS-ODN on the cultured human larynx carcinoma cell lines. Methods Total RNA was extracted from human laryngocarcinoma Hep-2 cells and the whole length gene of survivin was obtained by RT-PCR; the gene was cloned into pGEM-T-Easy vector and then sequenced. With the use of Hep-2 cells highly expressed survivin as the target cells and AS-ODN as a blocking agent, the inhibitory effect of AS-ODN was detected by MTT, so was the impact of AS-OND on survivin gene expression using RT-PCR. Results DNA sequencing showed that the sequence of the obtained gene was exactly the same as that reported in the GenBank. The growth of Hep-2 cells and the expression of survivin gene were suppressed by AS-ODN （P 〈 0.05）. Conclusions The survivin gene has been successfully cloned, which lays the foundation for further studies on the roles of survivin in the diagnosis and treatment of larynx carcinoma and other malignancies. Survivin AS-ODN may play a key role in the growth of larynx carcinoma cells and it may be a novel target of gene therapy for larynx carcinoma.