广藿香醇和广藿香酮治疗急性髓性白血病的药效研究

发布时间：2018-07-14 20:40

【摘要】：目的：探讨广藿香醇和广藿香酮诱导急性髓性白血病(acute myelocytic leukemia, AML)细胞MV4-11凋亡的机制,初步揭示二者治疗AML的作用。方法：1.采用四甲基偶氮唑盐(MTT)法检测广藿香醇和广藿香酮对12种共15株肿瘤细胞株及2种正常细胞株的体外抑制活性,计算半数抑制率(IC50),筛选敏感细胞株；2.25、50、100μg/mL的广藿香醇或广藿香酮作用于MV4-11细胞24h,采用Hoechst 33258染色技术和荧光显微成像系统观察细胞核形态变化；利用PI单染法和流式细胞计数仪检测MV4-11细胞周期；同时用流式细胞仪采用Annexin V-FITC/碘化丙啶(PI)双染法检测细胞凋亡率；3.采用western blot免疫印迹法检测广藿香醇对MV4-11细胞内NF-κB, PKM2及Caspase-3蛋白表达的影响；检测广藿香酮对MV4-11细胞内ERK信号通路相关蛋白和凋亡蛋白caspase-7表达的影响。结果：1.对多种肿瘤细胞株生长的影响：广藿香醇和广藿香酮在一定浓度下均能够有效抑制15株肿瘤细胞株的生长,IC50在19.6～100 μg/mL之间,其中对急性髓性白血病细胞株MV4-11最为敏感,IC50分别为23.2±4.9 μg/mL、22.6 ±3.7 μg/mL。2.对MV4-11细胞周期和凋亡的影响：25、50、100 μg/mL广藿香醇或广藿香酮作用MV4-11细胞后,在荧光显微镜下均能观察到细胞核皱缩,染色质密集和致密浓染的碎块；广藿香醇能够阻滞细胞周期于Gg2/M期,与溶剂对照组G2/M期细胞的41.6%相比,广藿香醇各剂量组G2/M期细胞数分别显著上升至48.7%(25 μg/mL)、55.1%(50μg/mL)和62.8%(100μg/mL),结果具有统计学差异(P0.05)；广藿香酮能阻滞MV4-11细胞周期于Go/G1期,与溶剂对照组Go/G1期细胞的33.2%相比,广藿香酮各剂量组Go/G1期细胞数分别显著上升至38%(25 μg/mL)、44.4%(50 μg/mL)和57.1%(100 μg/mL),结果具有统计学差异(P0.05)。Annexin V/PI双染流式细胞检测结果显示,25、50、100μg/mL广藿香醇和广藿香酮均能诱导MV4-11细胞发生凋亡,其中,广藿香醇组的凋亡率分别为8.9%、13.6%、22.0%,广藿香酮组分别为1.6%、10.1%、14.1%,与各组的溶剂对照组比较显著升高(P0.05)；3.对MV4-11细胞凋亡相关蛋白表达的影响：Western blot结果显示,在剂量50μg/mL时,广藿香醇明显抑制细胞内PKM2磷酸化蛋白的表达水平,其总蛋白的表达量不变；同时NF-κB和Caspase-3的表达量发生改变(P0.05)；在剂量25 μg/mL时,广藿香酮能够使急性髓性白血病细胞内的磷酸化ERK和Caspase-7发生改变(P0.05)。结论：(1)广藿香醇和广藿香酮能够抑制多种肿瘤细胞株的生长,但作用强度不同,其中对人急性髓性白血病细胞的作用最敏感；(2)广藿香醇和广藿香酮均可诱导白血病细胞MV4-11凋亡,广藿香醇阻滞细胞周期于G2/M期,广藿香酮阻滞细胞周期于Go/G1期；(3)广藿香醇诱导MV4-11细胞凋亡的机制是通过下调磷酸化PKM2和NF-κB蛋白量的表达,进一步激活caspase-3诱导凋亡的发生；广藿香酮诱导MV4-11细胞凋亡的机制是通过下调ERK的磷酸化激活caspase-7诱发凋亡。
[Abstract]:Aim: to investigate the mechanism of apoptosis induced by patchouli alcohol and patchoulone in acute myeloid leukemia (acute myelocytic leukemia,) cells. Method 1: 1. MTT assay was used to detect the inhibitory activity of patchouli alcohol and patchouli ketone on 12 tumor cell lines and 2 normal cell lines in vitro. The half inhibition rate (IC50) was calculated and sensitive cell lines were screened. The cell nuclear morphology was observed by Hoechst 33258 staining technique and fluorescence microscopic imaging system, and the cell cycle of MV 4-11 cells was detected by Pi single staining and flow cytometry, and the cell nuclear morphology was observed by Hoechst 33258 staining technique and fluorescence microscopic imaging system after treatment with 2. 25 渭 g / mL patchouli alcohol or patchoulone for 24 h. At the same time, the rate of apoptosis was detected by Annexin V-FITC / Pi double staining with flow cytometry. The effects of patchouli alcohol on the expression of NF- 魏 B, PKM2 and Caspase-3 in MV4-11 cells were detected by western blot Western blotting, and the caspase-7 expression of ERKsignaling pathway related proteins and apoptotic proteins in MV4-11 cells was detected by using patchouli ketone. The result is 1: 1. The effects on the growth of various tumor cell lines were as follows: patchouli alcohol and patchoulone could effectively inhibit the growth of 15 tumor cell lines in the range of 19.6 渭 g / mL and 100 渭 g / mL, respectively. The IC50 of MV4-11 cell line was 23.2 卤4.9 渭 g / mL ~ (-1) and 22.6 卤3.7 渭 g / mL ~ (2), respectively. Effects on cell cycle and apoptosis of MV4-11 cells treated with 1: 2550 100 渭 g / mL patchouli alcohol or patchoulone, nuclear shrinkage, dense chromatin and dense stained fragments were observed under fluorescence microscope. Patchouli alcohol could block the cell cycle in G _ 2 / M phase. Compared with the solvent control group, the cell number of G _ 2 / M phase increased significantly to 48.7% (25 渭 g / mL) 55.1% (50 渭 g / mL) and 62.8% (100 渭 g / mL) respectively, compared with 41.6% of the control group (P0.05). Patchouli ketone could block the cell cycle of MV4-11 cells at the G / G 1 phase, compared with 33. 2% of the cells in the G / G 1 phase of the solvent control group. The number of G / G 1 phase cells significantly increased to 38% (25 渭 g / mL) 44.4% (50 渭 g / mL) and 57.1% (100 渭 g / mL), respectively. The results were statistically different (P0.05). Annexin / VPI double staining flow cytometric analysis showed that 2550100 渭 g / mL patchouli alcohol and patchoulone could induce apoptosis of MV4-11 cells. The apoptotic rate of patchouli alcohol group was 8. 9% and that of patchouli group was 13. 6% and 22. 0, respectively, and that of patchouli group was 1. 6% and 10. 1% respectively, which was significantly higher than that of solvent control group (P 0. 05). The effect on the expression of apoptosis-related protein in MV4-11 cells showed that the expression of PKM2 phosphorylated protein was significantly inhibited by patchouli at a dose of 50 渭 g / mL, and the expression of total protein remained unchanged. At the same time, the expression of NF- 魏 B and Caspase-3 was changed (P0.05), and patchoulone could change phosphorylated ERK and Caspase-7 in acute myeloid leukemia cells at the dose of 25 渭 g / mL (P0.05). Conclusion: (1) patchouli alcohol and patchoulone can inhibit the growth of various tumor cell lines, but the action intensity is different, among which the effect on human acute myeloid leukemia cells is the most sensitive; (2) both patchouli alcohol and patchoulone could induce apoptosis of leukemia cell line MV4-11. Patchouli alcohol could block cell cycle at G 2 / M phase, and patchouli ketone block cell cycle at G / G 1 phase. (3) the mechanism of apoptosis induced by patchouli alcohol was that the expression of phosphorylated PKM2 and NF- 魏 B protein was down-regulated and the apoptosis induced by caspase-3 was further activated. Patchouli ketone induced apoptosis of MV4-11 cells by down-regulating phosphorylation of ERK and activating apoptosis by caspase-7.
【学位授予单位】：成都中医药大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R273

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