摘要
目的探讨川楝子提取物对白血病CEM细胞生长抑制作用及其机制。方法CEM细胞培养至对数生长期,采用不同浓度川楝子提取物(16μg/mL、80μg/mL、400μg/mL、2 mg/mL、10 mg/mL)进行干预,MTT检测细胞抑制率并推算半数抑制浓度;吖啶橙染色观察细胞结构改变;罗丹明123染色检测线粒体膜电位(ΔΨm)变化;流式细胞术检测细胞凋亡率和周期阻滞;免疫细胞化学检测Cyclin D1和CDK4蛋白表达;Western blot实验检测Bcl-XL、Bak、Cyt-C、Caspase-9和Caspase-3蛋白的表达。结果川楝子提取物能抑制CEM细胞的生长,在一定范围内对药物剂量和作用时间呈正向关联;吖啶橙染色可见给药后细胞核固缩及凋亡小体;川楝子提取物作用CEM细胞后,同对照组比较OD值显著增大(P<0.05或P<0.01),ΔΨm下降;流式细胞术检测给药组细胞凋亡率同对照组比较差异有显著统计学意义(P<0.01),随着药物浓度增加,G_(0)/G_(1)期细胞比例也逐渐加大,且细胞内Cyclin D1和CDK4表达逐渐减少,与对照组相比差异有显著统计学意义(P<0.01);各浓度川楝子提取物作用CEM细胞48 h后Cyt-C、Caspase-9和Caspase-3蛋白明显上调(P<0.05或P<0.01),Bcl-XL/Bak比值减小。结论川楝子提取物能抑制CEM细胞的增殖,其机制可能与以下两方面有关:①阻滞CEM细胞在G_(0)/G_(1)期;②抑制线粒体介导的凋亡途径中Bcl-XL蛋白的表达,同时上调Bak、Cyt-C、Caspase-9和Caspase-3蛋白的表达,从而促进CEM细胞发生凋亡。
Objective To investigate the inhibitory effect of Chuanlianzi(Toosendan Fructus)extract on the growth of leukemia CEM cells and its mechanism.Methods CEM cells were cultured to logarithmic growth stage and intervened with different concentrations of Chuanlianzi(Toosendan Fructus)extract(16μg/mL,80μg/mL,400μg/mL,2 mg/mL,10 mg/mL).The cell inhibition rate was detected by MTT and the half inhibition concentration was calculated.Acridine orange staining was used to observe the changes of cell structure.The apoptosis rate and cycle arrest were detected by flow cytometry.Rho123 staining was used to detect mitochondrial membrane potential(ΔΨm)changes.The expressions of cyclin D1 and CDK4 were detected by immunocytochemistry.The expressions of Bcl-XL,Bak,Cyt-C,Caspase-9 and Caspase-3 proteins were detected by Western blot.Results Chuanlianzi(Toosendan Fructus)extract can inhibit the growth of CEM cells,and has a positive correlation with drug dose and action time in a certain range.Acridine orange staining showed nuclear pyknosis and apoptotic bodies after administration.After treating CEM cells with toosendan extract,compared with those in the control group,OD value in the administration group increased significantly(P<0.05 or P<0.01)andΔΨm decreased.Compared with that in the control group,the apoptosis rate in the administration group was significantly different by flow cytometry(P<0.01).With the increase of drug concentration,the proportion of G_(0)/G_(1)cells gradually increased,and the expressions of Cyclin D1 and CDK4 in cells gradually decreased,which were significantly different from those in the control group(P<0.01).After 48 hours,the levels of Cyt-C,Caspase-9 and Caspase-3 proteins in CEM cells were significantly increased(P<0.05 or P<0.01),while Bcl-XL/Bak ratio decreased.Conclusion The extract of Chuanlianzi(Toosendan Fructus)can inhibit the proliferation of CEM cells,and its mechanism may be related to the following two aspects:①blocking CEM cells in G_(0)/G_(1)phase;②inhibiting the expression of Bcl XL protein in mitochondrial mediated apoptosis pathway,and up-regulating the expressions of Bak,Cyt-C,Caspase-9 and Caspase-3 proteins,thus promoting the apoptosis of CEM cells.
作者
朱大诚
刘振帅
吴慧婷
徐笑明
康林之
常娜
ZHU Dacheng;LIU Zhenshuai;WU Huiting;XU Xiaoming;KANG Linzhi;CHANG Na(Jiangxi University of Chinese Medicine,Nanchang 330004,Jiangxi,China)
出处
《中华中医药学刊》
CAS
北大核心
2023年第4期4-8,I0011,I0012,I0013,共8页
Chinese Archives of Traditional Chinese Medicine
基金
江西省“双一流”学科(中医学)建设基金项目(JXYLXK-ZHYI051)
江西省研究生创新专项基金项目(YC2020-B146)。
关键词
川楝子
白血病CEM细胞
周期阻滞
线粒体
凋亡
以毒攻毒
Chuanlianzi(Toosendan Fructus)
leukemia CEM cells
cycle block
mitochondrial
apoptosis
fighting toxin with toxin
