【摘要】 目的 探讨铁螯合剂去铁胺（DFO）对诱导白血病细胞HL-60的分子机制。 方法 2003年7-12月用钙黄绿素（calcein）检测HL-60细胞LIP。台盼蓝活细胞拒染实验进行活细胞计数及细胞存活率测定；光镜形态学观察及流式细胞仪（FCM）等方法检测HL-60细胞凋亡；比色法检测caspase-3（基于pNA标记底物的比色法）活性。 结果 ①不同浓度的DFO作用于HL-60细胞后，随培养时间延长及DFO浓度的增加，动态铁池降低，细胞生存率逐渐下降，凋亡率增加，显示一定的时间剂量依赖性。②HL-60细胞在不同浓度的DFO作用下，caspase-3的活性逐渐升高。50、100 μmol/L DFO作用于HL-60细胞24 h，caspase-3酶活性升高明显，与对照组相比，有统计学意义（Plt;0.001）；相关分析结果显示，HL-60细胞LIP的改变与caspase-3活性变化呈负相关系（r=-0.887，Plt;0.05）。 结论 DFO诱导白血病细胞凋亡的作用可能与螯合细胞内铁，降低细胞LIP，激活caspase-3，最终实施细胞凋亡密切相关。【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.
ObjectiveTo investigate whether desferrioxamine (DFO) can enhance the homing of bone marrow mesenchymal stem cells (BMSCs) and improve neovascularization in random flaps of rats.MethodsBMSCs and fibroblasts (FB) of luciferase transgenic Lewis rats were isolated and cultured. Forty 4-week-old Lewis male rats were used to form a 10 cm×3 cm rectangular flap on their back. The experimental animals were randomly divided into 4 groups with 10 rats in each group: in group A, 200 μL PBS were injected through retrobulbar venous plexus; in group B, 200 μL FB with a concentration of 1×106 cells/mL were injected; in group C, 200 μL BMSCs with a concentration of 1×106 cells/mL were injected; in group D, cells transplantation was the same as that in group C, after cells transplantation, DFO [100 mg/(kg·d)] were injected intraperitoneally for 7 days. On the 7th day after operation, the survival rate of flaps in each group was observed and calculated; the blood perfusion was observed by laser speckle imaging. Bioluminescence imaging was used to detect the distribution of transplanted cells in rats at 30 minutes and 1, 4, 7, and 14 days after operation. Immunofluorescence staining was performed at 7 days after operation to observe CD31 staining and count capillary density under 200-fold visual field and to detect the expressions of stromal cell derived factor 1 (SDF-1), epidermal growth factor (EGF), fibroblast growth factor (FGF), and Ki67. Transplanted BMSCs were labeled with luciferase antibody and observed by immunofluorescence staining whether they participated in the repair of injured tissues.ResultsThe necrosis boundary of ischemic flaps in each group was clear at 7 days after operation. The survival rate of flaps in groups C and D was significantly higher than that in groups A and B, and in group D than in group C (P<0.05). Laser speckle imaging showed that the blood perfusion units of flaps in groups C and D was significantly higher than that in groups A and B, and in group D than in group C (P<0.05). Bioluminescence imaging showed that BMSCs gradually migrated to the ischemia and hypoxia area and eventually distributed to the ischemic tissues. The photon signal of group D was significantly stronger than that of other groups at 14 days after operation (P<0.05). CD31 immunofluorescence staining showed that capillary density in groups C and D was significantly higher than that in groups A and B, and in group D than in group C (P<0.05). The expressions of SDF-1, EGF, FGF, and Ki67 in groups C and D were significantly stronger than those in groups A and B, and in group D than in group C. Luciferase-labeled BMSCs were expressed in the elastic layer of arteries, capillaries, and hair follicles at 7 days after transplantation.ConclusionDFO can enhance the migration and homing of BMSCs to the hypoxic area of random flap, accelerate the differentiation of BMSCs in ischemic tissue, and improve the neovascularization of ischemic tissue.