摘要
【目的】为探索XIAP相关因子1(Xaf1)调节肿瘤细胞凋亡的机制,利用基因开关调节系统(Tet-on),拟建立由doxycycline调控表达的Xaf1诱导细胞株。【方法】将pTREHA-Xaf1和pWZL-Hyg质粒用基因转染技术转入稳定表达rtTA的Saos-2细胞中。经过hygromycin的抗性筛选,挑出并扩增表达Xaf1的细胞株。在8例实验组中加入doxycycline,和不加doxycycline的8例对照组比较,重复3次实验用免疫印迹法和免疫荧光显微镜检测doxycycline对Xaf1表达的调控。Xaf1诱导的细胞凋亡由流式细胞检测DNA含量来表示。【结果】在30个抗hygromycin的细胞株中,免疫印迹法筛选出5个明显由doxycycline调控诱导表达Xaf1的细胞株,免疫荧光显微镜检测显示doxycycline诱导Xaf1表达于细胞核内。流式细胞检测Xaf1于8h开始诱导Saos细胞凋亡,凋亡率最高约20%,而且不影响细胞周期。【结论】Xaf1-Saos诱导细胞株是研究Xaf1调节肿瘤细胞凋亡机制的良好细胞模型;Xaf1是一种核蛋白,能独立诱导肿瘤细胞凋亡。
[Objective]To investigate the mechanism of apoptosis of tumor cells modulated by XIAP associate facter 1 （Xaf1）, we generated Xafl inducible cell lines, using Tet-on system, “gene switch” system, which can induce Xafl gene expression by administration of doxycycline. [Methods]Plasmids pTREHA-Xaf1 and pWZL-Hyg encoding hygromycin were co-transfected in the human osteosareoma Saos-2 cells, which express rtTA stably. Following hygromycin screening, the survived cells expressing Xafl were selected and enriched. Compared with the untreated cells （8 cases）, the regulating effect of doxycycline on the expression of Xafl gene in the treated cells （8 cases） was estimated respectively with Western blot analysis and immunofluoresces assay in three respective experiments. The effect of Xafl induction on apoptosis was measured by flow cytometry. [Results]From the 30 hygromycin-resistant cell strains, we selected 5 cell strains expressing Xafl in response to stimulation by doxycycline. Immunofluoresces assay showed that Xafl located in the nucleus. Flow cytometry demonstrated that apoptotic cells began to accumulate at 8 h,reached the peak of 20% and the cell cycle was not effected. [Conclusions]Xaf1-Saos inducible cell line is a good system to investigate the mechanism of apoptosis of tumor cells induced by Xafl. Xafl is a nuclear protein and can induce apoptosis alone without effecting cell cycling.
出处
《中山大学学报（医学科学版）》
CAS
CSCD
北大核心
2005年第5期528-532,共5页
Journal of Sun Yat-Sen University:Medical Sciences
基金
广东省卫生厅科研基金资助项目(E00d003012)
