靶向封闭survivin诱导喉癌细胞凋亡及抑制其增殖的体外实验

Induction of apoptosis and inhibition of proliferation in Hep-2 by survivin gene targeting in vitro

目的:探讨靶向抑制survivin表达对喉癌细胞凋亡和增殖的影响。方法:构建survivin反义RNA表达载体。用脂质体转染技术将重组质粒转染人喉癌细胞株Hep-2,利用G418(300g/L的维持浓度)筛选,获得稳定转染株(HpEGFP/survivin)。用Western-blot检测survivin反义RNA封闭survivin后蛋白表达效果。吖啶橙染色和流式细胞仪、四唑盐(MTT)和软琼脂集落形成实验分别检测HpEGFP/surivin的凋亡和增殖状态的改变,并与Hep-2和转染空载体的细胞(HpEGFP-C1)进行比较。结果:Western-blot结果表明,构建的survivin反义RNA表达载体部分抑制了Hep-2survivin蛋白的表达。转染反义survivinRNA载体的HpEGFP/survivin较Hep-2和HpEGFP-CI凋亡增多,其凋亡率较空载体对照组增加1.81倍,而其增殖力下降(P<0.01),软琼脂集落形成能力也明显下降(P<0.05)。结论:构建的survivin反义RNA表达载体部分抑制survivin蛋白的表达,并拮抗了survivin基因的抗凋亡作用,抑制喉癌细胞体外增殖能力和生存能力。

Objective：To study induction of apoptosis and inhibition of proliferation in Hep-2 by survivin gene targeting. Method：Antisense survivin RNA expression vector was constructed and then was transfected to human laryngeal carcinoma cell line Hep-2 by lipofectamine. HpEGFP/survivin cells were obstained by using G418. The level of survivin protein before and after transfection was determined by Western blot. Proliferation activity was measured by MTT assay. The experiment of colony formation in soft agar was carried out. Apoptosis was assessed by flow cytometry and acrdine orange（AO）. Result：After antisense survivin RNA plasmids were transfeeted, the level of survivin protein was inhibited in Hep-2. Compared with control, proliferation of HpEGFP/survivin cells transfected with the recombinant of antisense survivin RNA were suppressed significantly. The experiment of colony formation in soft agar showed the ability of colony formation decreased in HpEGFP/survivin cells compared to control（ P d0.05）. Apoptosis rate increased about 1.81 folds compared with control. Conclusion：The recombiton antisense survivin RNA plasmid can partly inhibit the level of survivin protein expression in Hep-2. Antisense survivin RNA can induce apoptosis and inhibit the proliferation of Hep-2 by down-regulating the expression of en- dogenous survivin in vitro.