反义Survivin对血管瘤内皮细胞增殖及凋亡的影响

目的 探讨脂质体转染Survivin反义寡核苷酸(ASODN)对体外培养的增生期血管瘤内皮细胞增殖、凋亡的影响.方法 人工合成Survivin ASODN,通过脂质体转染到体外培养的增生期血管瘤内皮细胞,光镜和倒置相差显微镜观察转染后细胞形态变化;用四氮唑盐(MTT)法观察转染后48 h不同浓度Survivin ASODN对细胞生长抑制的影响;RT-PCR、Western-blot及免疫组化法检测转染后内皮细胞Survivin mRNA水平、蛋白水平表达及Caspase-3蛋白表达.结果 不同浓度的Survivin ASODN作用后内皮细胞多数变圆、变小、脱落,胞膜皱褶、卷曲,细胞核固缩、碎裂,核被染成深蓝色或蓝黑色,以600 nmol/L作用72 h改变最明显.细胞生长受到明显抑制,600 nmol/LSurvivin ASODN时细胞生长抑制率最高,可达70.5%.与对照组相比,实验组Survivin mRNA、蛋白水平表达均明显下调,Caspase-3蛋白表达明显上调.结论 在体外RNA干扰靶向抑制Survivin基因可以显著抑制增生期血管瘤内皮细胞增殖,促进细胞凋亡;Survivin可能是控制血管瘤内皮细胞增殖或凋亡的关键基因之一.

Abstract：

Objective To evaluate the effects of survivin on cell proliferation and apoptosis in proliferative hemangioma endothelial cells. Methods Hemangioma endothelial cells were cultured in vitro and transfected with Survivin antisense oligonucleotides. The morphological changes were assessed with light microscopy and inverted phase contrast microscopy and electron microscopy. MTT assay was carried out to determine cell proliferation in proliferative hemangioma endothelial cells treated with antisense compounds. By using RT-PCR and Western blot, the expression levels of survivin mRNA and proteins were quantified. Active caspase-3 was evaluated by immunohistochemistry. Results Some morphological changes (e. g. round endothelial cells, reduced cell volume, folded cell membrane and condensed nuclei ) were revealed after transfection with increasing concentration of survivin ASODN, and these changes were most significant at the dose of 600nM at 72 hours. Endothelial cell growth was suppressed at the concentration of 600nmol/L Survivin ASODN. Survivin mRNA, protein were down-regulated significantly and Caspase-3 was up-regulated significantly in the experimental groups. Conclusions Down-regulation of survivin expression induced by the antisense compounds reduces cell growth potential, promotes apoptosis in proliferative hemangioma endothelial cells. Survivin protein is a key molecule associated with proliferation and apoptosis,and antisense oligonucleotides targeting survivin could be used as a potential therapy of proliferative hemangioma.

Survivin antisense compound inhibits proliferation and promotes apoptosis in proliferative hemangioma endothelial cells

Objective To evaluate the effects of survivin on cell proliferation and apoptosis in proliferative hemangioma endothelial cells. Methods Hemangioma endothelial cells were cultured in vitro and transfected with Survivin antisense oligonucleotides. The morphological changes were assessed with light microscopy and inverted phase contrast microscopy and electron microscopy. MTT assay was carried out to determine cell proliferation in proliferative hemangioma endothelial cells treated with antisense compounds. By using RT-PCR and Western blot, the expression levels of survivin mRNA and proteins were quantified. Active caspase-3 was evaluated by immunohistochemistry. Results Some morphological changes (e. g. round endothelial cells, reduced cell volume, folded cell membrane and condensed nuclei ) were revealed after transfection with increasing concentration of survivin ASODN, and these changes were most significant at the dose of 600nM at 72 hours. Endothelial cell growth was suppressed at the concentration of 600nmol/L Survivin ASODN. Survivin mRNA, protein were down-regulated significantly and Caspase-3 was up-regulated significantly in the experimental groups. Conclusions Down-regulation of survivin expression induced by the antisense compounds reduces cell growth potential, promotes apoptosis in proliferative hemangioma endothelial cells. Survivin protein is a key molecule associated with proliferation and apoptosis,and antisense oligonucleotides targeting survivin could be used as a potential therapy of proliferative hemangioma.