摘要
本研究旨在观察多西他赛对人骨髓瘤细胞系RPMI8226增殖和凋亡的影响,探讨其作用的分子机制。采用MTT法检测骨髓瘤细胞增殖抑制率,光学显微镜及电子显微镜观察多西他赛对骨髓瘤细胞形态的影响,Annex-in-ⅤFITC/PI双染流式细胞术检测细胞凋亡率,PI染色流式细胞术检测骨髓瘤细胞周期分布情况,半定量RT-PCR检测多西他赛对BCL-2、caspase-8及caspase-3 mRNA表达影响,Western blot法检测多西他赛作用前后骨髓瘤细胞BCL-2蛋白表达变化。结果显示:0.25-8.0μg/ml终浓度的多西他赛均可抑制RPMI8226细胞增殖,抑制率呈时间(r=0.927)和浓度(r=0.726)依赖性;多西他赛处理组光学显微镜下可见细胞数量明显减少,排列紊乱,高倍镜可见凋亡细胞,偶见坏死细胞;电子显微镜下可见细胞典型凋亡改变以及少数坏死细胞;多西他赛可明显诱导骨髓瘤细胞凋亡(P<0.01),主要将细胞阻滞于G2/M期(P<0.01),作用48 h时BCL-2 mRNA与BCL-2蛋白表达量减少(P<0.05),caspase-8、caspase-3 mRNA表达量增加(P<0.05)。结论:多西他赛通过诱导细胞凋亡可抑制骨髓瘤细胞增殖,其诱导细胞凋亡可能与细胞周期阻滞,激活细胞内、外源性凋亡通路有关。
This study was aimed to investigate the effects of docetaxel on proliferration and apoptosis of multiple myeloma cell line RPMI8226 and its mechanism. The inhibitory rate of multiple myeloma cells was detected by MTT, the morphological and ultrastrnctural changes of RPMI8226 cells were observed by using inverted fluorescent microscope and transmission electron microscope, the apoptosis-inducing effect of docetaxel on RPMI-8226 cells was determined by flow cytometry with Annexin-V F1TC/PI staining, the cell distritution in cell cycle of RPMI-8226 cells was assayed using flow cytometry with PI staining; the effect of docetaxel on expression of BCL-2, caspase-8, caspase-3 mRNA was detected by semiquantitative RT-PCR, the expression changes of BCL-2 protein in RPMI-8226 cells before and after treatment with docetaxel were measured by using Western blot. The results indicated that 0.25 - 8.0 μg/ml docetaxel obviously inhibited the proliferation of RPMI-8226 cells in both time- and dose-dependent manners. Cell number of docetaxel-treated group was significantly less than control group under inverted flurescent microscope, and the cell arrangement was irregular, necrotic cells could be seen occasionally. By transmission electron microscope, the morphological and ultrastructural changes of cell typical apoptosis could be observed, a few necrotic cells could be captured, too. Compared with control group, docetaxel induced the apoptosis of RPMI-8226 cell line （ P 〈 0.01 ）. Docetaxel mainly arrested RPMI-8226 cells in the G2/M phase（ P 〈0.01 ）. The expression of BCL-2 mRNA decreased （ P 〈 0. 05 ）, while the mRNA expression of caspase-8 and caspase-3 increased （ P 〈 0.05 ）. Western blot indicated that BCL-2 protein expression also decreased （ P 〈 0.05 ）. It is concluded that docetaxel can inhibit the proliferation of RPMI- 8226 cells by inducing cell apoptosis. Activation of the mitochondrial and death receptor pathways of apoptosis may be involved in the docetaxel-induced apoptosis, cell cycle arrest may also play an important role in the apoptosis mechanism.
出处
《中国实验血液学杂志》
CAS
CSCD
北大核心
2012年第6期1378-1383,共6页
Journal of Experimental Hematology
基金
西安交通大学医学院第一附属医院科研基金资助(编号201102)
