双向凝胶电泳分析三尖杉酯碱诱导K562细胞凋亡

Two-dimensional Polyacrylamide Gel Electrophoresis Analysis of Apoptosis in K562 Cells Induced by Harringtonine

背景与目的:三尖杉酯碱(harringtonine,HT)作为一种抗肿瘤药,已广泛用于治疗急、慢性髓系白血病,并已获得了良好的疗效。目前的研究表明其抗肿瘤作用与诱导肿瘤细胞凋亡有关,但具体分子机制不详。本研究主要是分析HT诱导K562细胞凋亡的蛋白谱,寻找凋亡相关蛋白。方法:应用AnnexinⅤ和PI双染法,通过流式细胞术区分HT诱导K562细胞凋亡早期和凋亡晚期阶段;进一步采用双向凝胶电泳技术和计算机辅助图像分析方法,对HT诱导凋亡的K562细胞与对照K562细胞进行分离和比较。结果:10μg/mlHT作用于K562细胞5h和24h,凋亡早期细胞(AnnexinⅤ+/PI-)分别为28.3%和18.1%(P<0.01),凋亡晚期细胞(AnnexinⅤ+/PI+)分别为9.1%和20.2%(P<0.01)。配比分析对照组细胞和凋亡早期组、晚期组细胞的蛋白图谱,统计分析表明:对照组K562细胞可分离出1300±50个蛋白点,组内蛋白点匹配率为(88.3±2.0)%。凋亡晚期组细胞中有10个蛋白点发生了明显和稳定的质和量的改变(P<0.01),其中8个蛋白点在凋亡后表达量上调,1个蛋白点表达下调,1个蛋白点只在对照细胞内表达。结论:差异表达的蛋白可能是三尖杉酯碱诱导K562细胞凋亡的相关蛋白。

BACKGROUND &OBJECTIVE: Harringtonine(HT),an anti-tumor drug, has been widely used to treat acute or chronic myeloid leukemia, and obtained satisf actory effects. Studies showed that the anti-tumor activity of HT is related wi th the apoptosis-inducing effect, but the molecular mechanisms remain unclear. This study was to analyze the protein maps contributed to the apoptosis in K562 cells induced by HT,and to screen the apoptosis-associated proteins. METHODS: F low cytometry was used to distinguish K562 cells of early apoptosis stage from t hose of late apoptosis stage through Annexin Ⅴand PI staining. Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE)was used to separate and compar e the HT-induced apoptotic K562 cells and control K562 cells. RESULTS: When K56 2 cells were treated with 10 靏/ml HT for 5,and 24 hours, percentages of early apoptotic cells (Annexin Ⅴ+/PI-)were 28.3%,and 18.1%(P< 0.01), while perc entages of late apoptotic cells (Annexin Ⅴ+/PI+)were 9.1%,and 20.2%(P< 0. 01), respectively. Statistical analysis showed (1 300±50) protein spots were re solved with a match rate of (88.3±2.0)%in control K562 cells. Ten protein spot s in late apoptotic cells displayed changes in expression after induction of HT for 24 hours (P< 0.01), of which 8 showed higher expression, 1 decreased,and 1 o nly expressed in control k562 cells. CONCLUSION: These proteins may be apoptosis-associated proteins of K562 cells induced by HT.