蟾蜍灵对人胶质瘤细胞U87MG及其成球细胞增殖和凋亡影响的研究  

Effect of Bufalin on Proliferation and Apoptosis of Human Glioma Cell Line U87MG and U87MG spheroids

作　　者：刘佳[1] 孔令凯 孙舒岚 李晓曦 张桂荣 朴浩哲 

机构地区：[1]辽宁中医药大学,辽宁沈阳110032 [2]大连医科大学肿瘤干细胞研究院,辽宁大连116044 [3]中国医科大学肿瘤医院辽宁省肿瘤医院中心实验室,辽宁沈阳110042

出　　处：《中国中医急症》2017年第11期1933-1937,共5页Journal of Emergency in Traditional Chinese Medicine

基　　金：辽宁省临床能力建设重大项目(LNCCC-B04-2015)

摘　　要：目的观察蟾蜍灵对人胶质瘤细胞系U87MG及U87MG成球细胞增殖和凋亡作用的影响。方法取对数生长期的U87MG细胞,制成细胞悬液,分别向细胞悬液中加入不同浓度的蟾蜍灵,另设DMSO作为对照组,MTT检测细胞存活情况;克隆形成2周后,观察不同浓度蟾蜍灵对细胞增殖的影响;蟾蜍灵处理U87MG细胞12 h和48 h后,流式细胞仪检测细胞凋亡情况;Western blot检测蟾蜍灵处理U87MG细胞48 h后凋亡相关蛋白Cleaved-caspase-3和Cleaved-PARP表达水平;分别用成球培养基和含有蟾蜍灵的成球培养基培养U87MG细胞7 d,显微镜下观察细胞是否成球及其形态、成球体积大小变化和数量变化;成球的U87MG细胞加入不同浓度蟾蜍灵72 h后,PI避光染色1 h,荧光显微镜下观察细胞死亡情况。结果加入蟾蜍灵处理的U87MG细胞生存率较空白对照组和DMSO组明显下降,24 h、48 h和72 h的IC50分别为85 nmol/L、34 nmol/L和22 nmol/L。蟾蜍灵处理后克隆形成数量低于空白对照组。U87MG细胞经蟾蜍灵处理后,细胞凋亡比例增加,并存在药物浓度时间依赖性;凋亡相关蛋白Cleaved-caspase-3和Cleaved-PARP表达量较空白对照组增加;U87MG细胞在成球培养基中培养后可以成球;加蟾蜍灵干预后,细胞成球体积明显比空白对照组小,且数量减少。PI染色观察到随药物浓度增加,细胞球死亡比例增加。结论蟾蜍灵可以抑制U87MG细胞生长和增殖,并能促进其凋亡;蟾蜍灵可以抑制胶质瘤U87MG成球,并能杀伤干细胞。Objective： To study the effects of bufalin on proliferation and apoptosis of human glioma cell line U87MG and U87MG spheroids. Methods： The logarithmic growth phase of U87MG cells were used to make cell suspensions. The cells were treated with different concentrations of Bufalin, and a DMSO control group was set up. Cell suivival level of U87MG was detected by MTF. The effect of different concentrations of bufalin on cell prolif- eration for 2 weeks was measured by colony formation. U87MG cells were treated with Bufalin for 12 and 48h,and apoptosis was assessed by flow cytometric analysis using Annexin V and propidium iodide （PI） double-staining. Western blot detected the expression of cleaved-caspase-3 and cleaved-PARP in U87MG cells after treated with Bufalin for 48 h. The U87MG cells were cultured in serum-free medium with or without Bufalin for 7 days. Then whether U87MG cells can be spheroids,the morphology of the cells,the change of the size and quantity of the spheroids were observed by microscope. U87MG spheroids were infected with Bufalin for 72h and stained with PI away from light for 1 hour. Subsequently,cells were visualized by fluorescence microscopy that red fluoreseencing ceils were indicative of cell death. Results： The survival rate of U87MG cells which treated with Bufalin were significantly decreased than the control group and DMSO group , and the IC50 of 24 h, 48 h and 72 h was respec- tively 85 nmol/L,34 nmol/L and 22 nmol/L. The number of clones treated with bufalin is lower than the control group ,and the apoptosis ratio increased with the concentration and time dependent. The expression of cleaved- caspase-3 and cleaved-PARP were higher than control group. U87MG cells maintained in serum-free mediumcan generate spheroids. The spheroids after treated with bufalin were significantly smaller than the control group, as well as the number of spheroids. PI staining was observed that cell death ratio increased with the increase of Bufalin＇s concentration. Conclusion： Bufalin can inh

关 键 词：蟾蜍灵 胶质瘤 凋亡 肿瘤干细胞 

分 类 号：R289.5[医药卫生—方剂学]