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<0.05)。结论苦参碱体外具有抑制 D341细胞增殖、诱导凋亡和促进自噬的作用。
目的：探討苦參堿在體外誘導人髓母細胞瘤 D341細胞增殖、凋亡和自噬的作用。方法體外培養細胞,隨機分為對照組、苦參堿0.5,1.0,1.5和2.0 g??L-1組,作用時間為24,48和72 h。 CCK-8法檢測細胞增殖,流式細胞儀檢測細胞凋亡,透射電鏡觀察細胞結構,Western蛋白質印跡法檢測細胞 Bax、Bcl-2、胱天蛋白酶3及自噬相關蛋白微管相關蛋白1輕鏈3(LC3)和Bcl-2同源結構域蛋白抗體beclin1蛋白錶達。隨後在苦參堿作用前1 h 加入終濃度為5 mmol??L-1的自噬抑製劑3-甲基腺嘌呤(3-MA),觀察細胞beclin1和 LC3蛋白錶達的變化。結果苦參堿0.5～2.0 g??L-1能明顯抑製 D341細胞增殖,具有濃度效應關繫(r24 h ＝0.994,r48 h ＝0.992,r72 h ＝0.996,P<0.01),而且可誘導 D341細胞凋亡( r24 h ＝0.937,r48 h ＝0.947,r72 h ＝0.987,P<0.01)；苦參堿濃度為2.0 g??L-1時,對 D341細胞增殖的抑製作用(r＝0.999,P<0.01)和凋亡的誘導作用(r＝0.990,P<0.01)具有時間效應關繫。透射電鏡觀察髮現,苦參堿2.0 g??L-1作用24 h, D341細胞齣現氣穴樣的空泡結構,細胞染色質濃縮、邊緣化；作用48 h,細胞覈染色質濃縮明顯,細胞胞漿中可見空泡；作用72 h,細胞覈固縮明顯,部分細胞可見覈裂解,空泡明顯增大。 Western蛋白質印跡法實驗結果錶明,苦參堿0.5～2.0 g??L-1增彊 D341細胞 Bax 蛋白錶達( r24 h ＝0.981,r48 h ＝0.967,r72 h ＝0.998, P<0.01),抑製 Bcl-2蛋白錶達(r24 h ＝-0.977,r48 h ＝-0.989, r72 h ＝-0.968,P<0.01)。苦參堿作用48 h 可增彊 D341細胞胱天蛋白酶3的錶達(r48 h ＝0.995,P<0.01),作用24,48和72 h能增彊 D341細胞 beclin1蛋白的錶達,具有濃度效應關繫(r24 h ＝0.989,r48 h ＝0.986,r72 h ＝0.966,P<0.01),自噬抑製劑3-MA 可抑製此作用(P<0.05)。苦參堿能使 D341細胞 LC3-Ⅰ蛋白錶達減少,LC3-Ⅱ錶達增加, LC3-Ⅰ/ LC3-Ⅱ比值降低(r24 h ＝-0.795,r48 h ＝-0.886,r72 h ＝-0.901,P<0.05)；自噬抑製劑3-MA 可抑製苦參堿對 LC3-Ⅰ和 LC3-Ⅱ蛋白錶達的影響(P<0.05)。結論苦參堿體外具有抑製 D341細胞增殖、誘導凋亡和促進自噬的作用。
목적：탐토고삼감재체외유도인수모세포류 D341세포증식、조망화자서적작용。방법체외배양세포,수궤분위대조조、고삼감0.5,1.0,1.5화2.0 g??L-1조,작용시간위24,48화72 h。 CCK-8법검측세포증식,류식세포의검측세포조망,투사전경관찰세포결구,Western단백질인적법검측세포 Bax、Bcl-2、광천단백매3급자서상관단백미관상관단백1경련3(LC3)화Bcl-2동원결구역단백항체beclin1단백표체。수후재고삼감작용전1 h 가입종농도위5 mmol??L-1적자서억제제3-갑기선표령(3-MA),관찰세포beclin1화 LC3단백표체적변화。결과고삼감0.5～2.0 g??L-1능명현억제 D341세포증식,구유농도효응관계(r24 h ＝0.994,r48 h ＝0.992,r72 h ＝0.996,P<0.01),이차가유도 D341세포조망( r24 h ＝0.937,r48 h ＝0.947,r72 h ＝0.987,P<0.01)；고삼감농도위2.0 g??L-1시,대 D341세포증식적억제작용(r＝0.999,P<0.01)화조망적유도작용(r＝0.990,P<0.01)구유시간효응관계。투사전경관찰발현,고삼감2.0 g??L-1작용24 h, D341세포출현기혈양적공포결구,세포염색질농축、변연화；작용48 h,세포핵염색질농축명현,세포포장중가견공포；작용72 h,세포핵고축명현,부분세포가견핵렬해,공포명현증대。 Western단백질인적법실험결과표명,고삼감0.5～2.0 g??L-1증강 D341세포 Bax 단백표체( r24 h ＝0.981,r48 h ＝0.967,r72 h ＝0.998, P<0.01),억제 Bcl-2단백표체(r24 h ＝-0.977,r48 h ＝-0.989, r72 h ＝-0.968,P<0.01)。고삼감작용48 h 가증강 D341세포광천단백매3적표체(r48 h ＝0.995,P<0.01),작용24,48화72 h능증강 D341세포 beclin1단백적표체,구유농도효응관계(r24 h ＝0.989,r48 h ＝0.986,r72 h ＝0.966,P<0.01),자서억제제3-MA 가억제차작용(P<0.05)。고삼감능사 D341세포 LC3-Ⅰ단백표체감소,LC3-Ⅱ표체증가, LC3-Ⅰ/ LC3-Ⅱ비치강저(r24 h ＝-0.795,r48 h ＝-0.886,r72 h ＝-0.901,P<0.05)；자서억제제3-MA 가억제고삼감대 LC3-Ⅰ화 LC3-Ⅱ단백표체적영향(P<0.05)。결론고삼감체외구유억제 D341세포증식、유도조망화촉진자서적작용。
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 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), and decrease the expression of Bcl-2 (r24 h = -0.977, r48 h = -0.989, r72 h = -0.968, P<0.01). Matrine could increase the expression of caspase 3 when the effect time was 48 h (r48 h =0.995, P<0.01). Matrine also increased the expression of beclin1 (r24 h =0.989, r48 h =0.986, r72 h =0.966, P<0.01). The autophagy inhibitor 3-MA could reduce this effect ( P < 0.05). Matrine decreased the expression of LC3-Ⅰ but increased the expression of LC3-Ⅱ and thus the ratio of LC3-Ⅰ/ LC-Ⅱ was decreased (r24 h = -0.795, r48 h = -0.886, r72 h = -0.901, P<0.05). 3-MA could reduce the effects of matrine on LC3-Ⅰ and LC3-Ⅱ expression of D341 cells (P<0.05). CONCLUSION Matrine can inhibit proliferation, induce apoptosis and promote autophagy of D341 cells in vitro.
