去甲斑蝥素体外干预复发性骨巨细胞瘤的效应与相关机制

摘要

Objective To investigate the mechanism of norcantharidin (NCTD) intervening the proliferation of recurrent giant cell tumor of bone (GCTB) in vitro.Methods GCTB tumor cells were isolated and cultured with NCTD at different concentrations (0,5,10,20,40,80 μg/mL) for in vitro intervention,and the intervention time points for each concentration group were 24 h,48 h,72 h,96 h,120 h,144 h,168 h,192 h.After intervention,cell viability was measured through CCK-8 assay,and the survival rate of GCTB tumor cells was calculated.The 0 μg/mL group,the 5 μg/mL group and the 10 μg/mL group were selected for the following experiments,and the blank group (saline medium) was established at the same time.Cell morphology was observed under electron microscope.The apoptosis rate of GCTB tumor cells was analyzed by flow cytometry.The expression change of related factors in receptor activator of nuclear factor-κB ligand (RANKL)/ receptor activator of nuclear factor-κB (RANK)/osteoprotegerin (OPG) signaling pathway were detected by real-time fluorescence reverse transcription -polymerase chain reaction (RT-PCR),western blot and immunohistochemistry.Results The results of CCK-8 assay showed that the survival rate of the GCTB tumor cells decreased with the increase of NCTD concentration (≤80 μtg/mL).When the concentration was above 10 μg/mL,the survival rate of GCTB tumor cells was significantly lower than that of the 0 μtg/mL group.The lowest survival rate of each concentration group was noted at 48 h.Flow cytometry analysis showed that the apoptotic rate of the blank group,the 0μg/mL group,the 5 μg/mL group and the 10 μg/mL group was (0.37±0.06)％,(4.65±0.94)％,(9.33±0.70)％ and (11.53±0.40)％,respectively (F=192.468,P =0.000).The results of RT PCR,Western blot and immunohistochemistry showed that NCTD inhibited the expression of osteoclast-derived factors [RANKL,tumor necrosis factor (TNF)α,macrophage colony-stimulating factor (M-CSF),interleukin (IL)6,matrix metalloproteinase (MMP)-2,MMP 9] in the RANKL/RANK/OPG signaling pathway,while the expression of OPG was not significantly inhibited.Conclusion NCTD could effectively inhibit the proliferation of recurrent GCTB probably through regulating the RANKL/RANK/ OPG signal transduction pathway.%目的 探究去甲斑蝥素(NCTD)对复发性骨巨细胞瘤(GCTB)细胞的体外干预效应及相关机制.方法 体外分离培养GCTB肿瘤细胞后,采用不同浓度的NCTD(0、5、10、20、40、80 μg/mL)进行体外干预,每个浓度组的干预时间点为24 h、48 h、72 h、96 h、120 h、144 h、168 h、192 h.干预后利用CCK-8法检测细胞活力,并计算细胞存活率.选取NCTD0、5、10 μg/mL组进行后续实验,并设立空白组(加入生理盐水的培养基).电镜下观察以上各组的细胞形态,应用流式细胞仪分析各组细胞的凋亡率,采用实时荧光定量逆转录-聚合酶链反应(RT-PCR)法、蛋白质免疫印迹法和免疫组化等方法检测各组细胞中破骨细胞分化因子(RANKL)/核因子-κB受体活化因子(RANK)/骨保护素(OPG)信号转导通路相关因子表达水平的变化.结果 CCK-8法检测结果显示,NCTD浓度≤80 μg/mL时,GCTB肿瘤细胞存活率随浓度增加而降低;当NCTD浓度＞10 μg/mL时,细胞存活率显著降低(与NCTD0 μg/mL组相比);各浓度组干预时间为48 h时细胞存活率最低.流式细胞分析结果显示,空白组及NCTD 0μg/mL组、5 μg/mL组、10 μg/mL组的细胞凋亡率分别为(0.37±0.06)％、(4.65±0.94)％、(9.33±0.70)％、(11.53±0.40) ％(F=192.468,P=0.000).RT-PCR法、蛋白质免疫印迹法和免疫组化的结果显示,NCTD对RANKL/RANK/OPG信号转导通路中的破骨源性因子(RANKL、肿瘤坏死因子-α、巨噬细胞集落刺激因子、白细胞介素-6、细胞基质金属蛋白酶-2、细胞基质金属蛋白酶-9)的表达有一定的抑制作用,对破骨细胞分化抑制因子(如OPG)的表达无显著的抑制作用.结论 NCTD对复发性GCTB中肿瘤细胞具有抑制作用,其作用可能是通过调节RANKL/RANK/OPG信号转导通路产生的.