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目的:探讨广藿香醇(PA)诱导MV4-11细胞凋亡,并探讨其可能存在的相关活性。方法:采用四甲基偶氮唑盐比色(MTT)法检测PA对人结、直肠癌细胞HCT116,人肝癌细胞Hep G-2,人肺癌细胞A549,人急性淋巴髓单核细胞白血病细胞MV4-11,人皮肤黑色素瘤细胞A375,小鼠乳腺癌细胞4T1,人单核细胞型淋巴瘤细胞THP-1,人正常胚肾293A细胞的增殖抑制作用;采用Hoechst 33258染色法观察细胞凋亡的形态学变化;通过Annexin V-FITC/碘化丙啶(PI)双染法并应用流式细胞术检测细胞凋亡;免疫印迹法(Western blot)检测磷酸化M2型丙酮酸激酶(p-PKM2),核转录因子-κB(NF-κB),半胱氨酸天冬氨酸蛋白酶3(Caspase-3)凋亡蛋白的表达。结果:PA能抑制人HCT116,Hep G-2,A549,MV4-11,A375,4T1,THP-1,293A细胞的增殖,半抑制浓度(IC50)分别为0.26,0.32,0.28,0.09,0.37,0.23,0.24,0.33 mmol·L-1;作用于MV4-11细胞24 h后可使细胞核皱缩,染色质凝聚,形成明显的凋亡小体。0.1,0.2,0.5 mmol·L-1PA能诱导MV4-11细胞凋亡,凋亡率分别为7.9%,13.6%,22.0%,与溶剂组比较,结果具有统计学差异(P<0.05);Western blot检测显示,PA使细胞NF-κB,p-PKM2,Caspase-3蛋白表达发生明显改变(P<0.05)。结论:PA能抑制人白血病细胞MV4-11细胞的增殖,并诱导其发生凋亡,作用机制可能与NF-κB,p-PKM2,Caspase-3蛋白量的改变有关。
Objective: To investigate the effects of patchoulol (PA) on the apoptosis of MV4-11 cells and to explore its possible related activities. Methods: MTT assay was used to detect the effect of PA on human colorectal cancer cells HCT116, human hepatoma Hep G-2, human lung adenocarcinoma A549, human acute lymphoblastic leukemia cell line MV4 -11, human skin melanoma A375, mouse breast cancer cell 4T1, human monocytic lymphoma THP-1 and normal human embryonic kidney 293A cells. Hoechst 33258 staining was used to observe the apoptosis Morphological changes were detected by flow cytometry. Annexin V-FITC / propidium iodide (PI) double staining and flow cytometry were used to detect the apoptosis. Phosphorylated M2 pyruvate kinase (p-PKM2 ), Nuclear factor kappa B (NF-κB) and caspase-3. Results: PA inhibited the proliferation of human HCT116, Hep G-2, A549, MV4-11, A375, 4T1 and THP-1, 293A cells with IC50 of 0.26,0.32,0.28,0.09,0.37,0.23 , 0.24,0.33 mmol·L-1. After treated with MV4-11 for 24 h, the nucleus was collapsed and chromatin was aggregated to form obvious apoptotic bodies. The apoptosis rates of MV4-11 cells were significantly increased at 0.1,0.2 and 0.5 mmol·L-1PA levels (7.9%, 13.6% and 22.0%, respectively) compared with the solvent group (P <0.05) The results of Western blot showed that the expression of NF-κB, p-PKM2 and Caspase-3 in PA significantly changed (P <0.05). Conclusion: PA can inhibit the proliferation of human leukemia cell line MV4-11 and induce its apoptosis. The mechanism may be related to the changes of the protein levels of NF-κB, p-PKM2 and Caspase-3.