突变型Survivin逆转HeLa细胞恶性表型的研究

Survivin Mutants Reverse the Malignancy of HeLa Cells

背景与目的:在大多数肿瘤组织中都发现了survivin的异常高表达现象,这说明survivin在肿瘤发生中具有重要作用。本文的目的在于研究突变型survivin对肿瘤细胞恶性表型的影响,并初步探讨其可能机制。方法:构建突变型survivin的表达载体,转染HeLa细胞,通过G418筛选,得到稳定表达的细胞克隆;利用流式细胞仪分析HeLa细胞的凋亡;通过Westernblot检测突变型survivin对细胞周期蛋白的影响。结果:突变型survivin表达质粒在HeLa细胞中表达良好,利用相应的抗体可以检测到其表达的蛋白;表达突变型survivin的HeLa细胞与正常对照相比,克隆形成能力明显减弱;瞬时转染突变型survivin的HeLa细胞可以自主发生凋亡;而且,突变型survivin还可以使多核HeLa细胞增多,survivin-N端的作用比survivinT34A更明显;突变型survivin可以影响周期蛋白的cyclinD1,使其蛋白水平分别下降了68%和12%。结论:突变型survivin可以部分逆转HeLa细胞的恶性表型,cyclinD1蛋白水平下降可能是其发挥作用的重要机制之一。

BACKGROUND &OBJECTIVE: Survivin was aberrantly expressed in most cancer tissues, suggesting that survivin plays an important role in carcinogenesis. This study was designed to investigate the function and mechanism of survivin mutants in tumor cells. METHODS: The site mutant and truncated survivin mutants were transfected into HeLa cells and selected using G418. Cell apoptosis was analyzed using flow cytometry. Protein level of cyclin D1 was detected by Western blot analysis. RESULTS: Survivin mutant plasmid expressed in the HeLa cells successfully. The expressed protiein could be detected using related antibody. Colony formation ability significantly decreased in the HeLa cells with survivin mutants compared with that in the parental HeLa cells. The HeLa cells transfected instantly with survivin mutants could undergo apoptosis automatically. Meanwhile, survivin mutants could cause an increase of mutlinuclear HeLa cells. The effect of survivin N showed more effective than that of survivin T34A. Survivin N and survivin T34A could influence the expression of cyclin D1 and reduced its protein levels of 68%and 12%, respectively. CONCLUSION: Survivin mutants can partially reverse the malignancy of HeLa cells. The reduction of cyclin D1 induced by survivin mutants may play an important role in it. Survivin may be a target gene in gene therapy of cancer.