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  

Mechanism of norcantharidin intervening the proliferation of recurrent giant cell tumor of bone in vitro

作　　者：王进 陈飞雁 夏军 王思群 魏亦兵 吴建国 黄钢勇 陈杰 石晶晟 杨元庆 WANG Jin;CHEN Feiyan;YANG Yuanqing;WANG Siqun;WEI Yibing;WU Jianguo;HUANG Gangyong;CHEN Jie;SHI Jingsheng;YANG Yuanqing(Department of Orthopaedics,Huashan Hospital Affiliated to Fudan University,Shanghai 200040,China)

机构地区：[1]复旦大学附属华山医院骨科,上海200040

出　　处：《国际骨科学杂志》2018年第2期93-99,共7页International Journal of Orthopaedics

基　　金：上海市卫生和计划生育委员会科研课题(201440585)

摘　　要：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、80μg/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、10μg/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≤80μg/mL时,GCTB肿瘤细胞存活率随浓度增加而降低;当NCTD浓度>10μg/mL时,细胞存活率显著降低(与NCTD0μg/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NCTD0μg/mL组、5μg/mL组、10μg/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。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(0,5,10,20,40,80μg/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 0μg/mL group,the 5μg/mL group and the 10μg/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(≤80μg/mL).When the concentration was above 10μg/mL,the survival rate of GCTB tumor cells was significantly lower than that of the 0μg/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 5μg/mL group and the 10μg/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 O

关 键 词：骨巨细胞瘤 去甲斑蝥素 靶向治疗 破骨细胞分化因子 

分 类 号：R738.1[医药卫生—肿瘤]