發(fā)布時(shí)間：2018-07-14 20:40

【摘要】：目的：探討廣藿香醇和廣藿香酮誘導(dǎo)急性髓性白血病(acute myelocytic leukemia, AML)細(xì)胞MV4-11凋亡的機(jī)制,初步揭示二者治療AML的作用。方法：1.采用四甲基偶氮唑鹽(MTT)法檢測(cè)廣藿香醇和廣藿香酮對(duì)12種共15株腫瘤細(xì)胞株及2種正常細(xì)胞株的體外抑制活性,計(jì)算半數(shù)抑制率(IC50),篩選敏感細(xì)胞株；2.25、50、100μg/mL的廣藿香醇或廣藿香酮作用于MV4-11細(xì)胞24h,采用Hoechst 33258染色技術(shù)和熒光顯微成像系統(tǒng)觀察細(xì)胞核形態(tài)變化；利用PI單染法和流式細(xì)胞計(jì)數(shù)儀檢測(cè)MV4-11細(xì)胞周期；同時(shí)用流式細(xì)胞儀采用Annexin V-FITC/碘化丙啶(PI)雙染法檢測(cè)細(xì)胞凋亡率；3.采用western blot免疫印跡法檢測(cè)廣藿香醇對(duì)MV4-11細(xì)胞內(nèi)NF-κB, PKM2及Caspase-3蛋白表達(dá)的影響；檢測(cè)廣藿香酮對(duì)MV4-11細(xì)胞內(nèi)ERK信號(hào)通路相關(guān)蛋白和凋亡蛋白caspase-7表達(dá)的影響。結(jié)果：1.對(duì)多種腫瘤細(xì)胞株生長(zhǎng)的影響：廣藿香醇和廣藿香酮在一定濃度下均能夠有效抑制15株腫瘤細(xì)胞株的生長(zhǎng),IC50在19.6～100 μg/mL之間,其中對(duì)急性髓性白血病細(xì)胞株MV4-11最為敏感,IC50分別為23.2±4.9 μg/mL、22.6 ±3.7 μg/mL。2.對(duì)MV4-11細(xì)胞周期和凋亡的影響：25、50、100 μg/mL廣藿香醇或廣藿香酮作用MV4-11細(xì)胞后,在熒光顯微鏡下均能觀察到細(xì)胞核皺縮,染色質(zhì)密集和致密濃染的碎塊；廣藿香醇能夠阻滯細(xì)胞周期于Gg2/M期,與溶劑對(duì)照組G2/M期細(xì)胞的41.6%相比,廣藿香醇各劑量組G2/M期細(xì)胞數(shù)分別顯著上升至48.7%(25 μg/mL)、55.1%(50μg/mL)和62.8%(100μg/mL),結(jié)果具有統(tǒng)計(jì)學(xué)差異(P0.05)；廣藿香酮能阻滯MV4-11細(xì)胞周期于Go/G1期,與溶劑對(duì)照組Go/G1期細(xì)胞的33.2%相比,廣藿香酮各劑量組Go/G1期細(xì)胞數(shù)分別顯著上升至38%(25 μg/mL)、44.4%(50 μg/mL)和57.1%(100 μg/mL),結(jié)果具有統(tǒng)計(jì)學(xué)差異(P0.05)。Annexin V/PI雙染流式細(xì)胞檢測(cè)結(jié)果顯示,25、50、100μg/mL廣藿香醇和廣藿香酮均能誘導(dǎo)MV4-11細(xì)胞發(fā)生凋亡,其中,廣藿香醇組的凋亡率分別為8.9%、13.6%、22.0%,廣藿香酮組分別為1.6%、10.1%、14.1%,與各組的溶劑對(duì)照組比較顯著升高(P0.05)；3.對(duì)MV4-11細(xì)胞凋亡相關(guān)蛋白表達(dá)的影響：Western blot結(jié)果顯示,在劑量50μg/mL時(shí),廣藿香醇明顯抑制細(xì)胞內(nèi)PKM2磷酸化蛋白的表達(dá)水平,其總蛋白的表達(dá)量不變；同時(shí)NF-κB和Caspase-3的表達(dá)量發(fā)生改變(P0.05)；在劑量25 μg/mL時(shí),廣藿香酮能夠使急性髓性白血病細(xì)胞內(nèi)的磷酸化ERK和Caspase-7發(fā)生改變(P0.05)。結(jié)論：(1)廣藿香醇和廣藿香酮能夠抑制多種腫瘤細(xì)胞株的生長(zhǎng),但作用強(qiáng)度不同,其中對(duì)人急性髓性白血病細(xì)胞的作用最敏感；(2)廣藿香醇和廣藿香酮均可誘導(dǎo)白血病細(xì)胞MV4-11凋亡,廣藿香醇阻滯細(xì)胞周期于G2/M期,廣藿香酮阻滯細(xì)胞周期于Go/G1期；(3)廣藿香醇誘導(dǎo)MV4-11細(xì)胞凋亡的機(jī)制是通過(guò)下調(diào)磷酸化PKM2和NF-κB蛋白量的表達(dá),進(jìn)一步激活caspase-3誘導(dǎo)凋亡的發(fā)生；廣藿香酮誘導(dǎo)MV4-11細(xì)胞凋亡的機(jī)制是通過(guò)下調(diào)ERK的磷酸化激活caspase-7誘發(fā)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 鹵4.9 渭 g / mL ~ (-1) and 22.6 鹵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.
【學(xué)位授予單位】：成都中醫(yī)藥大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R273

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