纳米雄黄抗B细胞非霍奇金淋巴瘤Raji细胞的体外作用研究

Effects of realgar nanoparticles on B cell non-Hodgkin's lymphoma Raji cells in vitro

目的考察纳米雄黄对B细胞非霍奇金淋巴瘤Raji细胞的体外作用。方法利用激光粒度仪、TEM和AFM对纳米雄黄和水飞雄黄进行表征;利用光镜、AFM和TEM依次观察纳米雄黄和水飞雄黄作用下Raji细胞的增殖形态变化、单细胞表面细胞膜变化以及细胞内超微结构的变化;MTT法检测纳米雄黄和水飞雄黄作用下的细胞存活率;利用荧光显微镜及流式细胞术观察纳米雄黄和水飞雄黄引起Raji细胞的凋亡和细胞周期分布情况。结果纳米雄黄的粒径为(79±8)nm,水飞雄黄的粒径为(1.89±0.2)μm。光镜下,可明显观察到纳米雄黄可抑制Raji细胞的聚集生长状态,AFM下可观察到纳米雄黄作用下的Raji细胞皱缩,体积变小,膜表面的黏附物质不再向四周伸展,而水飞雄黄作用下的Raji细胞变化不明显。TEM下可观察到纳米雄黄作用下的Raji细胞胞内亚细胞器受到破坏,线粒体空泡明显增多,水飞雄黄组变化不明显。MTT结果显示,50 mg·L^(-1)纳米雄黄作用Raji细胞24 h时,细胞的存活率为(40±2)%,而相同剂量的水飞雄黄作用组为(65±3)%;50 mg·L^(-1)纳米雄黄作用Raji细胞48 h时,Raji细胞的存活率仅为10%,而相同剂量的水飞雄黄组,Raji细胞的存活率为(42±2)%。荧光显微镜下可观察到纳米雄黄作用下的Raji细胞核凋亡明显,水飞雄黄组作用不明显。流式细胞术结果显示,水飞雄黄作用下的Raji细胞总凋亡率为11.14%,而纳米雄黄处理组的Raji细胞总凋亡率为15.9%。与水飞雄黄相比,纳米雄黄作用下Raji细胞在G_1期的分布比例明显升高,S期分布比率下降。结论与水飞雄黄相比,相同剂量的纳米雄黄在相同作用时间下,可明显抑制B细胞淋巴瘤Raji细胞的增殖,破坏其亚细胞结构,进而引起其凋亡。

AimTo observe the effects of realgar nanoparticles on B cell non Hodgkin’s lymphoma Raji cells in vitro.MethodsRealgar nanoparticles and crude realgar particles were characterized with a laser particle size analyzer,a transmission electron microscopy(TEM)and an atomic force microscopy(AFM).The morphological changes of proliferation of Raji cells brought about by the use of realgar naoparticles and crude realgar particles were observed with a light microscope.The membrane changes of Raji cells treated with realgar naoparticles and crude realgar particles were observed with AFM.The ultrastructures of Raji cells were observed with TEM.The inhibitory effects of Raji cells treated with realgar naoparticles and crude realgar particles were measured with MTT.The nuclear apoptosis morphologies of Raji cells were observed with fluorescence microscopy.The apoptosis rates and the cell cycle distributions of Raji cells treated with realgars were measured with flow cytometry.ResultsThe size of realgar nanoparticles and crude realgar particles was(79±8)nm and(189±02)μm,respectively.Light microscopy showed that realgar nanoparticles could inhibit the aggregation growth of Raji cells.AFM showed that Raji cells treated with realgar nanoparticle became shrank,had smaller volume and lost the growth state of stretching out.Raji cells treated with crude realgars did not change significantly.TEM showed Raji cells treated with realgar nanoparticle had damaged subcellular organelles and mitochondria with increased vacuoles.The Raji cells treated with crude realgar did not change significantly.MTT assay showed that when treated with the final concentration of50mg·L-1of realgar nanoparticle for24h,the cell survival rate of Raji cells was(40±2)%.When treated with the same concentration of crude realgar,its survival rate was(65±3)%.When treated with50mg·L-1of realgar nanoparticle for48h,its survival rate was only10%,and when treated with crude realgar,its survival rate was(42±2)%.Fluorescence microscope indicated that the Raji cells treated with realgar nanoparticle had obvious nuclear apoptosis,which was not obvious in crude realgar group.Flow cytometry showed that the total apoptosis rate of Raji cells induced by realgar nanoparticles and by crude realgar was1114%,159%,respectively.Compared with those treated with crude realgar,the Raji cells treated with realgar nanoparticles presented a significantly higher ratio cell distribution in G1phase and an obvious decreased ratio in S phase.ConclusionCompared with crude realgar particles,the same dose of realgar nanoparticles can significantly inhibit the proliferation of Raji cells,destroy their sub cellular structure,and induce the cell apoptosis of Raji cells.