摘要
目的探讨苦参碱在体外诱导人髓母细胞瘤D341细胞增殖、凋亡和自噬的作用。方法体外培养细胞,随机分为对照组、苦参碱0.5,1.0,1.5和2.0 g·L-1组,作用时间为24,48和72 h。CCK-8法检测细胞增殖,流式细胞仪检测细胞凋亡,透射电镜观察细胞结构,Western蛋白质印迹法检测细胞Bax、Bcl-2、胱天蛋白酶3及自噬相关蛋白微管相关蛋白1轻链3（LC3）和Bcl-2同源结构域蛋白抗体beclin1蛋白表达。随后在苦参碱作用前1 h加入终浓度为5 mmol·L-1的自噬抑制剂3-甲基腺嘌呤（3-MA）,观察细胞beclin1和LC3蛋白表达的变化。结果苦参碱0.5~2.0 g·L-1能明显抑制D341细胞增殖,具有浓度效应关系（r24 h=0.994,r48 h=0.992,r72 h=0.996,P〈0.01）,而且可诱导D341细胞凋亡（r24 h=0.937,r48 h=0.947,r72 h=0.987,P〈0.01）;苦参碱浓度为2.0 g·L-1时,对D341细胞增殖的抑制作用（r=0.999,P〈0.01）和凋亡的诱导作用（r=0.990,P〈0.01）具有时间效应关系。透射电镜观察发现,苦参碱2.0 g·L-1作用24 h,D341细胞出现气穴样的空泡结构,细胞染色质浓缩、边缘化;作用48 h,细胞核染色质浓缩明显,细胞胞浆中可见空泡;作用72 h,细胞核固缩明显,部分细胞可见核裂解,空泡明显增大。Western蛋白质印迹法实验结果表明,苦参碱0.5~2.0 g·L-1增强D341细胞Bax蛋白表达（r24 h=0.981,r48 h=0.967,r72 h=0.998,P〈0.01）,抑制Bcl-2蛋白表达（r24 h=-0.977,r48 h=-0.989,r72 h=-0.968,P〈0.01）。苦参碱作用48 h可增强D341细胞胱天蛋白酶3的表达（r48 h=0.995,P〈0.01）,作用24,48和72 h能增强D341细胞beclin1蛋白的表达,具有浓度效应关系（r24 h=0.989,r48 h=0.986,r72 h=0.966,P〈0.01）,自噬抑制剂3-MA可抑制此作用（P〈0.05）。苦参碱能使D341细胞LC3-Ⅰ蛋白表达减少,LC3-Ⅱ表达增加,LC3-Ⅰ/LC3-Ⅱ比值降低（r24 h=-0.795,r48 h=-0.886,r72 h=-0.901,P〈0.05）;自噬抑制剂3-MA可抑制苦参碱对LC3-Ⅰ和LC3-Ⅱ蛋白表达的影响（P〈0
OBJECTIVE To explore the effect of matrine induced proliferation, apoptosis and auto-phagy on human medulloblastoma cell line D341 in vitro. METHODS D341 cells in vitro were incubated＆amp;nbsp;with matrine 0, 0.5, 1.0, 1.5 and 2.0 g.L-1 for 24, 48 and 72 h, respectively. The proliferation of D341 cells was analyzed using Cell Counting Kit-8 assay. Apoptosis was detected by flow cytometry. The mor-phologic change of cells was observed under a transmission electron microscope. The expression of Bax, Bcl-2, caspase 3, microtubule associated protein 1 light chain 3 （LC3） and beclin1 was detected by Western blotting, and the expression of LC3 and beclin1 was detected by Western blotting with or without the autophagy inhibitor 3-methyladenine（3-MA）. 3-MA was added 1 h before matrine and the final concentration of 3-MA was 5 mmol.L-1 . RESULTS Matrine significantly inhibited the proliferation of D341 cells. There was a concentration-effect relationship （ r24 h = 0.994, r48 h = 0.992, r72 h = 0.996, P〈0.01）. Matrine could induce the cell apoptosis （r24 h = 0.937, r48 h = 0.947, r72 h = 0.987, P〈0.01）. When the concentration of matrine was 2.0 g.L-1 , the inhibitory effect on D341 cell proliferation （r=0.999, P〈0.01） and the induction of cell apoptosis （r=0.990, P〈0.01） had a time-dependence. When the concen-tration of matrine was 2.0 g.L-1 , the ultrastructure of the D341 cells had obvious change. Cells with acoustic cavitation bubble structure, chromatin condensation, and marginalization were observed after matrine treatment for 24 h. After 48 h treatment with matrine, nuclear chromatin condensation and more vacuoles in the cytoplasm were observed. After 72 h treatment with matrine, cells exhibited apoptotic characteristics with obvious nuclear chromatin condensation, and nuclear fragmentation, significantly increased the larger cytoplasmic vacuoles. Western blotting analysis showed that matrine could increase the expression of Bax （r24 h =0.981, r48 h =0.967, r72 h =0.998, P〈0.01�
出处
《中国药理学与毒理学杂志》
CAS
CSCD
北大核心
2015年第2期240-246,共7页
Chinese Journal of Pharmacology and Toxicology
基金
浙江省中医药管理局科技计划资助项目(2013ZA133)~~
关键词
苦参碱
细胞增殖
细胞凋亡
自噬
matrine
cell proliferation
apoptosis
autophagy
