• 1 鄭州市兒童醫(yī)院血液科（鄭州，450003);2 四川大學(xué)華西第二醫(yī)院血液腫瘤實(shí)驗(yàn)室;

【摘要】 目的  探討鐵螯合劑去鐵胺（DFO）對(duì)誘導(dǎo)白血病細(xì)胞HL-60的分子機(jī)制。 方法  2003年7-12月用鈣黃綠素（calcein）檢測(cè)HL-60細(xì)胞LIP。臺(tái)盼藍(lán)活細(xì)胞拒染實(shí)驗(yàn)進(jìn)行活細(xì)胞計(jì)數(shù)及細(xì)胞存活率測(cè)定；光鏡形態(tài)學(xué)觀察及流式細(xì)胞儀（FCM）等方法檢測(cè)HL-60細(xì)胞凋亡；比色法檢測(cè)caspase-3（基于pNA標(biāo)記底物的比色法）活性。 結(jié)果  ①不同濃度的DFO作用于HL-60細(xì)胞后，隨培養(yǎng)時(shí)間延長(zhǎng)及DFO濃度的增加，動(dòng)態(tài)鐵池降低，細(xì)胞生存率逐漸下降，凋亡率增加，顯示一定的時(shí)間劑量依賴性。②HL-60細(xì)胞在不同濃度的DFO作用下，caspase-3的活性逐漸升高。50、100 μmol/L DFO作用于HL-60細(xì)胞24 h，caspase-3酶活性升高明顯，與對(duì)照組相比，有統(tǒng)計(jì)學(xué)意義（P lt;0.001）；相關(guān)分析結(jié)果顯示，HL-60細(xì)胞LIP的改變與caspase-3活性變化呈負(fù)相關(guān)系（r=-0.887，P lt;0.05）。 結(jié)論  DFO誘導(dǎo)白血病細(xì)胞凋亡的作用可能與螯合細(xì)胞內(nèi)鐵，降低細(xì)胞LIP，激活caspase-3，最終實(shí)施細(xì)胞凋亡密切相關(guān)。
【Abstract】 Objective  To observe the changes of caspase-3 activity during apoptosis of HL-60 cells induced by an iron deferoxamine (DFO). Methods  Exponentially growing HL-60 cells (1×106/mL) were used in this experiment from July 2003 to December 2003. The study groups were divided as follows: DFO group, iron+DFO group and control group. The viability was detected by typanblue, apoptosis was assessed by morphological study and flow cytometry (FCM) assay, and the caspase-3 activity was detected by melorimetry. The intracellular label iron pool (LIP) was measured with a fluorimetric assay using the metalsensitive probe calcein-AM. Results  ①When HL-60 cells were incubated with different concentrations of DFO, viability assay was lower than that in the control group at the 12th, 24th and 48th hour (P lt;0.05). ② The cells incubated with different concentrations of DFO showed dose-time dependence and was much higher than that in the control group (P lt;0.01). ③The caspase-3 activity was significantly higher in the apoptotic cells than that in the control cells. Conclusions  The apoptosis of HL-60 cells induced by DFO may be correlated with the decrease of cellular LIP and activity of caspase-3.

引用本文： 賈國(guó)存,李豐益,高舉. 鐵螯合劑誘導(dǎo)白血病細(xì)胞凋亡中caspase-3的變化. 華西醫(yī)學(xué), 2010, 25(9): 1683-1685. doi: 復(fù)制

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5. Epsztejn S, Kakhlon O, Glickstein H, et al. Fluorescence analysis of the labile iron pool of mammalian cells[J]. Anal Biochem, 1997, 248(1): 31-40.
6. Palozza P, Serini S, Torsello A, et al. Mechanism of activation of caspase cascade during beta-carotene-induced apoptosis in human tumor cells[J]. Nutr Cancer, 2003, 47(1): 76-87.
7. Keri G, Racz G, Magyar K, et al. Pro-apoptotic and anti-apoptotic molecules affecting pathways of signal transduction[J]. Ann NY Acad Sci, 2003, 1010(12): 109-112.
8. Roy S, Bayly CI, Gareau Y, et al. Maintenance of caspase-3 proenzyme dormancy by an intrinsic “safety catch” regulatory tripeptide[J]. Proc Natl Acad Sci USA, 2001, 98(11): 6132-6137.
9. 黃貴清, 廖清奎, 羅春華. 急性白血病患兒外周血白血病細(xì)胞轉(zhuǎn)鐵蛋白受體表達(dá)分析[J]. 華西醫(yī)科大學(xué)學(xué)報(bào), 1997, 28(1): 55-57.
10. 賈國(guó)存, 湯有才, 李豐益, 等. HL-60細(xì)胞鐵池變化與凋亡相關(guān)基因關(guān)系的初步研究[J]. 中國(guó)小兒血液與腫瘤雜志, 2007, 5(12): 209-212.
11. 劉玉峰, 王叨, 張傳新. 去鐵胺誘導(dǎo)白血病細(xì)胞HL-60凋亡線粒體膜電位變化的研究[J]. 中華兒科雜志, 2005, 43(4): 300-301.
12. Fang D, Bao Y, Li X, Liu F. Effects of iron deprivation on multidrug resistance of leukemic K562 cells[J]. Chemotherapy, 2010, 56(1): 9-16.
13. Noulsri E, Richardson DR, Lerdwana S. et al. Antitumor activity and mechanism of action of the iron chelator, Dp44mT, against leukemic cells[J]. Am J Hematol, 2009, 84(3): 170-176.
14. Mori S, Sawada T, Okada T, et al. Anti-proliferative effect of interferon-gamma is enhanced by iron chelation in colon cancer cell lines in vitro[J]. Hepatogastroenterology, 2008, 55(85): 1274-1279.
15. Kontoghiorghes GJ, Efstathiou A, Ioannou-Loucaides S, et al. Chelators controlling metal metabolism and toxicity pathways: applications in cancer prevention, diagnosis and treatment[J]. Hemoglobin, 2008, 32(1-2): 217-227.
  1. 1. Meggiato T, Calabrese F, De Cesare CM, et al. C-JUN and CPP32 (CASPASE 3) in human pancreatic cancer: relation to cell proliferation and death[J]. Pancreas, 2003, 26(1): 65-70.
  2. 2. Roy S, Bayly CI, Gareau Y, et al. Maintenance of caspase-3 proenzyme dormancy by an intrinsic “safety catch” regulatory tripeptide[J]. Proc Natl Acad Sci USA, 2001, 98(11): 6132-6137.
  3. 3. 劉玉峰, 賈國(guó)存, 曾利, 等. 鐵剝奪誘導(dǎo)HL-60細(xì)胞凋亡及對(duì)化療藥物誘導(dǎo)HL-60細(xì)胞凋亡的影響[J]. 中華兒科雜志, 2001, 39(12): 735-738.
  4. 4. Yang R, Muller C, Huynh V, et al. Functions of cyclin A1 in the cell cycle and its interactions with transcription factor E2F-1 and the Rb family of proteins[J]. Mol Cell Biol, 1999, 19(3): 2400-2407.
  5. 5. Epsztejn S, Kakhlon O, Glickstein H, et al. Fluorescence analysis of the labile iron pool of mammalian cells[J]. Anal Biochem, 1997, 248(1): 31-40.
  6. 6. Palozza P, Serini S, Torsello A, et al. Mechanism of activation of caspase cascade during beta-carotene-induced apoptosis in human tumor cells[J]. Nutr Cancer, 2003, 47(1): 76-87.
  7. 7. Keri G, Racz G, Magyar K, et al. Pro-apoptotic and anti-apoptotic molecules affecting pathways of signal transduction[J]. Ann NY Acad Sci, 2003, 1010(12): 109-112.
  8. 8. Roy S, Bayly CI, Gareau Y, et al. Maintenance of caspase-3 proenzyme dormancy by an intrinsic “safety catch” regulatory tripeptide[J]. Proc Natl Acad Sci USA, 2001, 98(11): 6132-6137.
  9. 9. 黃貴清, 廖清奎, 羅春華. 急性白血病患兒外周血白血病細(xì)胞轉(zhuǎn)鐵蛋白受體表達(dá)分析[J]. 華西醫(yī)科大學(xué)學(xué)報(bào), 1997, 28(1): 55-57.
  10. 10. 賈國(guó)存, 湯有才, 李豐益, 等. HL-60細(xì)胞鐵池變化與凋亡相關(guān)基因關(guān)系的初步研究[J]. 中國(guó)小兒血液與腫瘤雜志, 2007, 5(12): 209-212.
  11. 11. 劉玉峰, 王叨, 張傳新. 去鐵胺誘導(dǎo)白血病細(xì)胞HL-60凋亡線粒體膜電位變化的研究[J]. 中華兒科雜志, 2005, 43(4): 300-301.
  12. 12. Fang D, Bao Y, Li X, Liu F. Effects of iron deprivation on multidrug resistance of leukemic K562 cells[J]. Chemotherapy, 2010, 56(1): 9-16.
  13. 13. Noulsri E, Richardson DR, Lerdwana S. et al. Antitumor activity and mechanism of action of the iron chelator, Dp44mT, against leukemic cells[J]. Am J Hematol, 2009, 84(3): 170-176.
  14. 14. Mori S, Sawada T, Okada T, et al. Anti-proliferative effect of interferon-gamma is enhanced by iron chelation in colon cancer cell lines in vitro[J]. Hepatogastroenterology, 2008, 55(85): 1274-1279.
  15. 15. Kontoghiorghes GJ, Efstathiou A, Ioannou-Loucaides S, et al. Chelators controlling metal metabolism and toxicity pathways: applications in cancer prevention, diagnosis and treatment[J]. Hemoglobin, 2008, 32(1-2): 217-227.