Extracellular traps released by neutrophils (neutrophil extracellular traps, NETs) are a double-edged sword, and understanding the mechanism of NET formation is of great significance for disease treatment. However, the short lifespan, the large individual differences, and the inability to perform gene editing render it difficult to decipher NET formation using neutrophils. It is necessary to find a model cell to replace neutrophils to study the mechanism of NET formation. In this study, we used different concentrations (0, 0.1, 1, and 10 μmol/L) of all-trans retinoic acid (ATRA) to differentiate HL-60 cells for different days (1, 3, 5, and 7 days). By detecting the cell viability and nuclear morphology of cells, we confirmed that HL-60 cells were differentiated to neutrophil-like cells (dHL-60) after treated with ATRA for at least 5 days. Using immunofluorescence staining to detect the formation of NETs, we demonstrated that dHL-60 cells differentiated for 5 days with 1 μmol/L ATRA could generate NETs comparable to those produced by neutrophils upon phorbol 12-myristate 13-acetate (PMA) stimulation, without histone H3 citrullination. Furthermore, the formation of NETs by dHL-60 cells were NADPH-dependent and PAD4-independent, consistent with neutrophils. Taken together, these observations suggest that dHL-60 cells differentiated with 1 μmol/L ATRA for 5 days can be used as a model cell for neutrophils to study the mechanism of NET formation.
【摘要】 目的 探討鐵螯合劑去鐵胺（DFO）對誘導白血病細胞HL-60的分子機制。 方法 2003年7-12月用鈣黃綠素（calcein）檢測HL-60細胞LIP。臺盼藍活細胞拒染實驗進行活細胞計數及細胞存活率測定；光鏡形態學觀察及流式細胞儀（FCM）等方法檢測HL-60細胞凋亡；比色法檢測caspase-3（基于pNA標記底物的比色法）活性。 結果 ①不同濃度的DFO作用于HL-60細胞后，隨培養時間延長及DFO濃度的增加，動態鐵池降低，細胞生存率逐漸下降，凋亡率增加，顯示一定的時間劑量依賴性。②HL-60細胞在不同濃度的DFO作用下，caspase-3的活性逐漸升高。50、100 μmol/L DFO作用于HL-60細胞24 h，caspase-3酶活性升高明顯，與對照組相比，有統計學意義（Plt;0.001）；相關分析結果顯示，HL-60細胞LIP的改變與caspase-3活性變化呈負相關系（r=-0.887，Plt;0.05）。 結論 DFO誘導白血病細胞凋亡的作用可能與螯合細胞內鐵，降低細胞LIP，激活caspase-3，最終實施細胞凋亡密切相關。【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 (Plt;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 (Plt;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.
