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目的:观察低浓度哇巴因对人白血病细胞株Jurkat生长的影响并初步探讨哇巴因特异性调节Jurkat细胞生长的机制。方法:分别用不同低浓度哇巴因作用于人白血病细胞株Jurkat后,采用四甲基偶氮唑盐MTT法检测细胞增殖情况、流式细胞学FCM技术检测细胞凋亡情况以及细胞内线粒体膜电位变化情况,Western blot法观察哇巴因对Jutkat细胞膜表面钠钾ATP酶的表达调节作用。结果:MTT及FCM检测结果表明随着哇巴因浓度的增高,哇巴因对Jurkat细胞的增殖抑制及促凋亡作用越明显。WesternBlot结果显示30nM及50nM哇巴因作用于Jurkat细胞株48h后引起细胞钠钾ATP酶表达下调,[3H]-哇巴因结合实验结果显示在Jurkat细胞株随着哇巴因作用浓度升高,细胞膜钠泵对哇巴因的亲和力逐渐下降。结论:低浓度哇巴因即可抑制白血病细胞株Jurkat增殖并诱导其凋亡。这种特异性细胞生长调控作用与哇巴因引起的细胞膜钠钾ATP酶表达变化相关,最终引起细胞内线粒体膜电位发生变化,释放相关凋亡蛋白,诱导细胞凋亡。
OBJECTIVE: To observe the effect of low concentration ouabain on the growth of human leukemia Jurkat cells and to investigate the mechanism of ouabain-specific regulation of Jurkat cell growth. Methods: After different concentrations of ouabain were applied to Jurkat cell line, MTT assay was used to detect the cell proliferation. Flow cytometry FCM was used to detect the cell apoptosis and mitochondrial membrane potential Potential changes were observed by Western blot ouabain Jutkat cell membrane surface sodium and potassium ATPase expression regulation. Results: The results of MTT and FCM showed that ouabain could inhibit the proliferation and induce the apoptosis of Jurkat cells more obviously with the increase of ouabain concentration. Western Blot results showed that 30nM and 50nM ouabain induced Jurkat cells 48h after down-regulated expression of sodium and potassium ATPase, [3H] - ouabain binding experiments showed that in Jurkat cell line with ouabain concentration increased, The affinity of the membrane sodium pump for ouabain decreased gradually. CONCLUSION: Low concentration of ouabain inhibits the proliferation and induces apoptosis of leukemia Jurkat cells. This specific cell growth regulation and ouabain-induced changes in cell membrane Na-K ATPase expression changes, eventually leading to changes in mitochondrial membrane potential, the release of the relevant apoptotic proteins, induce apoptosis.