款冬花粗多糖体外诱导人白血病K562细胞的凋亡

Apoptosis of K562 cells induced by Flos Farfarae polysaccharide in vitro

目的:观察款冬花粗多糖对体外培养人白血病K562细胞有无凋亡诱导作用。方法:实验于2006-06-25/2006-09-08在河北北方学院实验中心细胞生物学实验室、分子生物学实验室、药物研究实验室完成。①款冬花粗多糖的提取:称取已干燥粉粹的款冬花417.0g,加入1000mL体积分数0.85乙醇回流提取1h,滤过,滤渣加入1000mL三蒸水回流提取2h,过滤,滤渣再加入1000mL三蒸水回流提取2h,合并2次滤液,减压浓缩至适量,四倍体积无水乙醇醇沉,静置过夜,过滤,沉淀依次用无水乙醇、丙酮、石油醚洗涤,即得款冬花粗多糖。苯酚-硫酸法测定款冬花粗多糖含量以生药计算为122.9mg。制成102.5g/L粗多糖溶液。②人白血病K562细胞的生长抑制实验:体外培养的K562细胞经10,30,50,70,90mg/L的款冬花粗多糖作用48h后,通过Hoeehst33258染色在荧光显微镜下进行形态学观察、采用琼脂糖凝胶电泳及流式细胞术检测细胞凋亡。结果:①经款冬花粗多糖作用后,荧光显微镜下发现体外培养的K562细胞中出现核固缩、凋亡小体。②经不同质量浓度款冬花粗多糖处理的K562细胞DNA电泳均出现相差约200bp的DNA梯子状条带。③流式细胞仪检测发现,经不同质量浓度款冬花粗多糖处理的K562细胞,有凋亡峰。结论:款冬花粗多糖可诱导体外培养人白血病细胞K562凋亡。

AIM： To investigate whether the Flos Farfarae polysacchadde can induce apoptosis of human K562 in vitro or not. METHODS： The experiment was performed at the Laboratory of Cell Biology, Laboratory of Molecular Biology and Laboratory of Medicine Research of Experimental Center, Hebei North University from June 25^th to September 8^th, 2006. ①extraction of Flos Farfarae polysaccharide： Weighted 417.0 g dried Flos Farfarae, the optimum extraction process was as follows： the content of alcohol was 0.85 volume fraction, 1 000 mL, 1 hour; extracted again with triple distrilled water 1 000 mL for the marc two times and put the extractions together. Concentrated it with water and alcohol, overnight, treated it with alcohol, acetone and soxhlet. The method of sulpuric acid-phenol was applied to determine the content of Flos Farfarae polysaccharide as 122.9 mg. And got the polysaccharide solution 102.5 g/L. ②Growth inhibiting K562 cells： K562 cultured in vitro, which were treated for 48 hours with different concentrations （10,30,50,70,90 mg/L）of Flos Farfarae polysaccharide, were detected through following ways： the changes of cell morphology were observed under fluorescence microscope with Hoeehst33258 staining; agrose electrophoresis and flow cytometry were used to detect apoptosis. RESULTS： ①K562 cells treated with Flos Farfarae polysaccharide presented cell contraction and apoptotic bodies under fluorescence microscope. ②200 bp DNA fragment appeared through agrose electrophoresis following FIos Farfarae polysaccharide of different concentrations. ③Flow cytometry determined that apoptotic peak appeared in K562 cells after Flos Farfarae polysaccharide of different concentrations. CONCLUSION： Flos Farfarae bolysaccharide can induce K562 cell aboptosis in vitro.