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目的对比As2O3不同干预方式对白血病细胞株NB4的侵袭力和基质金属蛋白酶2,9(MMP-2、MMP-9)活性的影响。方法噻唑蓝(MTT)法检测As2O3恒定和变化两种体系体外干预对NB4细胞的增殖抑制,ELISA检测2种体系中处理的NB4细胞的MMP-2和MMP-9蛋白含量的变化。明胶酶谱法检测2种As2O3处理前后NB4细胞MMP-2活性变化,Westen blot检测活性MMP-9蛋白表达,Transwell小室穿透实验检测两种As2O3处理方式对NB4细胞穿透力的影响。结果①与平行对照组比较0.1μmol·L-1As2O3恒定浓度体系中持续作用72h,对MMP-2、MMP-9的活性和NB4细胞侵袭力影响不显著。0.5μmol·L-1As2O3持续作用24h与对照组比较无明显差异,48h以后MMP-2、MMP-9的活性和NB4侵袭力降低,与对照组比较差异显著。1.0μmol·L-1以上浓度As2O3处理24h,MMP-2、MMP-9活性和NB4细胞侵袭力下降,并呈时间依赖性。②2.0μmol·L-1As2O3恒定浓度体系与峰浓度为5.0μmol·L-1,终质量浓度为0μmol·L-1的变化浓度体系比较,前者对NB4的侵袭力和MMPs活性的抑制程度比后者更显著。结论NB4细胞的侵袭力与其MMP-2、MMP-9活性有关,促分化有效的低浓度As2O3对NB4细胞侵袭力和MMP-2、MMP-9活性抑制作用不明显。在干预时间相同的情况下,促凋亡有效的恒定浓度As2O3持续作用,对NB4细胞侵袭力和MMP-2、MMP-9活性的抑制强度大于峰浓度很高但持续时间很短的变化浓度体系,这可能是临床上As2O3持续缓慢输注法较常规给药法更能有效降低急性早幼粒细胞白血病(APL)中枢神经系统浸润和早期的致死性脑出血发生率的机制之一。
Objective To compare the invasiveness of leukemic cell line NB4 and the activity of matrix metalloproteinase 2, 9 (MMP-2, MMP-9) with different interventions of As2O3. Methods Thiazolyl blue (MTT) assay was used to detect the inhibitory effect of As2O3 in vitro on the proliferation of NB4 cells. The levels of MMP-2 and MMP-9 in NB4 cells treated with 2 kinds of As2O3 were detected by ELISA. The activity of MMP-2 in NB4 cells was measured by gelatin zymography before and after treatment with two kinds of As2O3. The expression of active MMP-9 protein was detected by Westen blot. The effects of two As2O3 treatments on the penetration of NB4 cells were detected by Transwell chamber-penetrating assay. Results ① Compared with parallel control group, the effect of 0.1μmol·L-1As2O3 constant concentration for 72h had no significant effect on the activity of MMP-2 and MMP-9 and the invasiveness of NB4 cells. After treated with 0.5μmol·L-1As2O3 for 24 hours, the activity of MMP-2 and MMP-9 and the invasiveness of NB4 were not significantly different from those of the control group at 48h, which was significantly different from the control group. The concentration of 1.0μmol·L-1 As2O3 treated 24h, MMP-2, MMP-9 activity and invasiveness of NB4 cells decreased in a time-dependent manner. ② Compared with the system of 2.0μmol·L-1As2O3 constant concentration system, the concentration of 5.0μmol·L-1 and the final concentration of 0μmol·L-1 changed the inhibitory ratio of NB4 invasiveness and MMPs activity More significant. Conclusions The invasiveness of NB4 cells is related to the activity of MMP-2 and MMP-9. The low effective concentration of As2O3 can inhibit the invasiveness of NB4 cells and the activity of MMP-2 and MMP-9. In the condition of the same intervention time, the sustained effect of constant concentration of As2O3, which is effective in promoting apoptosis, inhibited the invasiveness of NB4 cells and the activity of MMP-2 and MMP-9 more than those of high concentration but short duration , Which may be one of the mechanisms by which slow and continuous infusion of As2O3 in clinical practice is more effective than the conventional administration method to reduce the incidence of central nervous system infiltration and early fatal cerebral hemorrhage in acute promyelocytic leukemia (APL).