【作者】 吴建国；

【导师】 夏大静；

【作者基本信息】 浙江大学 ， 免疫学， 2008， 硕士

【摘要】 目的:研究大萼香茶菜甲素(MA)抑制白血病细胞株HL-60增殖,诱导细胞分化和凋亡作用,并对其作用机制进行初步探讨。方法:将不同浓度的MA与HL-60细胞在体外培养,台盼蓝染色、MTT比色法观察对HL-60细胞增殖的抑制作用;细胞形态学、DNA含量、DNA梯度电泳及细胞周期分析、Annexin-V/PI双标记和Hoechst 33258荧光染色等分析细胞凋亡;通过细胞表面抗原CD11b、CD13、CD14,NBT试验和细胞形态学检测MA对HL-60细胞的分化作用;流式细胞术检测Bcl-2、Bax、P53、Fas和线粒体膜蛋白、线粒体跨膜电位(Δψm)的表达变化;RT-PCR检测Bcl-2、Bax、P53、caspase-3 mRNA变化。结果:MA呈时效及量效性地抑制HL-60细胞的增殖和活力,8μg/ml MA作用HL-60细胞24～72h后,MTT法检测24h、48h、72h的细胞增殖抑制率分别为(52.9±0.3)%、(70.0±1.0)%和(75.3±1.2)%,均显著高于对照组(P＜0.01);24h、48h、72h的IC50分别为8.76、7.17、7.14μg/ml;HL-60细胞经MA作用后,大部分细胞阻滞于G0/1期,DNA片段化,出现典型的细胞形态改变,经4～20μg/ml的MA作用后,SubG1由(2.85±1.6)%上升至(51.7±5.9)%,与对照组(1.74±0.5)%相比具有显著性差异(P＜0.05),Annexin-V/PI标记升高,Hoechst染色后的凋亡细胞的特征性改变等均表明MA能诱导HL-60细胞凋亡;HL-60细胞经4-8μg/ml MA作用24h后,细胞发生部分分化,细胞表面CD11b表达增加,NBT阳性细胞增多。MA诱导HL-60细胞凋亡和分化过程中,Bcl-2表达无变化,但Bax表达显著增加,Bax/Bcl-2的比值升高,Fas、P53表达无变化。随MA作用的浓度增加,caspase-3表达升高、线粒体膜电位(Δψm)下降、而线粒体膜蛋白表达显著升高。结论:MA能抑制HL-60细胞增殖和细胞活力、诱导HL-60细胞向粒系分化、促进细胞凋亡,其机制与上调bax基因和bax/bcl-2比值、caspase-3激活与线粒体膜电位显著下降等有关。更多还原

【Abstract】 Objective:To study the effect of MA on proliferation inhibition, differentiation and apoptosis in HL-60 human leukemia cell line and explore its possible mechanisms.Methods:Different concentration of MA and the different time of cultivation were used to treat HL-60 cell.The proliferation inhibition was analyzed by Trypan blue staining and MTr assay.Cell apoptosis was analyzed by cell morphology,DNA agarose gel electrophoresis,DNA content and cell cycle analyzation,Annexin-Ⅴ/PI,Hoechst 33258 fluorescence staining.The cell morphological analysis,expression of CD11b,CD13,CD14,were performed to evaluate differentiation of HL-60 cells.The expressions of bcl-2,bax,Fas, P53 and mitochondrial membrane protein were analyzed by flow cytometry, While the mitochondrial transmembrancepotential(ΔΨm) was labeled by dihydrorhodamin 123.RT-PCR method was used to study the bcl-2、bax、p53、caspase-3 mRNA levels.Results:MA could inhibit HL-60 cell proliferation viability within a certain range of treating time and doses,with a 24h IC50 of 8.76μg/ml,48 h of 7.17μg/ml and 72h of 7.14μg/ml.A majority of HL-60 cells were arrested in G0/G1 phase.The HL-60 cells apoptosis was confirmed by type cell morphology,DNA fragment,sub-G1 phase and Annexin-Ⅴ/PI Labeling method with a time and dose related manner.The morphology of HL-60 cells cultured in the presence of 4-8μg/ml MA for 16hs was more mature with higher positive rate of NBT and up-regulated expressions of CD11b than those cultured without MA.The expression of bax was increased,and bcl-2、p53、fas were unchanged by the treatment of MA.MA could increase the expression of mitochondrial membrane protein and caspase-3 in a dose-dependent manner while theΔΨm was reduced.Conclusion::MA can inhibit proliferation,induce differentiation and apoptosis of HL-60 cells.The mechanism may associate with its up-regulation of bax,open the mitochondrial permeabilitytransition pore and reduceΔΨm.更多还原