摘要
目的探讨新型免疫抑制剂FC-007对胶质母细胞瘤(GBM)增殖、凋亡和侵袭的影响及其分子机制。方法体外培养胶质母细胞瘤细胞株U87MG和U118MG,细胞计数试剂盒(CCK-8)检测不同浓度FC-007(0、0.1、0.5、1、5、10、50、100 μmol/L)处理胶质瘤细胞后的细胞增殖活性。应用流式细胞术(FCM)检测不同浓度FC-007(0、10、20 μmol/L)对U87MG和U118MG细胞周期和凋亡的影响。应用蛋白质印迹法(Western blot)检测不同浓度FC-007(0、25、50、100 μmol/L)对基质金属蛋白酶(MMP)-2、MMP-9蛋白表达的影响,实时定量反转录聚合酶链反应(RT-qPCR)法检测FC-007不同浓度(0、5、10、20 μmol/L)对Nrf2及其下游基因HO-1、NQO-1表达水平的影响。组间比较采用F检验。结果 CCK-8结果显示,FC-007作用48 h后U87MG和U118MG细胞株增殖抑制浓度(IC50)分别为9.77、11.10 μmol/L,FCM结果显示,随FC-007浓度增加,处于G0/G1期的细胞比例显著高于空白对照组,处于S期的细胞比例显著低于空白对照组,G2/M期无明显改变,差异均有统计学意义(F=121.962、125.478、129.653、120.516、129.340、122.425,P<0.05);FC-007干预胶质瘤细胞24 h后,在0、10、20 μmol/L浓度下,细胞凋亡率分别为,U87MG细胞(6.41±0.35)%、(18.43±1.27)%、(26.11±1.74)%;U118MG细胞(5.02±0.75)%、(28.49±5.60)%、(33.35±4.12)%,差异均有统计学意义(F=265.502、260.378,P<0.05)。Western blot结果显示,FC-007显著降低MMP-2、MMP-9蛋白的表达,差异均有统计学意义(F=178.835、182.098、175.229、186.427,P<0.05)。RT-qPCR结果显示,FC-007可抑制GBM细胞中Nrf2及其下游基因HO-1及NQO-1的表达,差异均有统计学意义(F=185.998、187.835、182.642、188.517、187.556、182.475,P<0.05)。结论 FC-007可降低MMP-2、MMP-9蛋白水平,降低Nrf2、HO-1、NQO-1的mRNA表达,以剂量时间依赖关系抑制胶质母细胞瘤细胞的增殖,促进细胞凋亡,并可将细胞周期阻滞在G0/G1期。
Objective To explore the effect of a new immunosuppressant FC-007 on the proliferation,apoptosis and invasion of glioblastoma(GBM)and its molecular mechanism.Methods Glioblastoma cell lines U87MG and U118MG were cultured in vitro,and cell counting kit-8(CCK-8)assay was used to detect the proliferation activity of glioma cells treated with different concentrations of FC-007(0,0.1,0.5,1,5,10,50,100μmol/L).Flow cytometry(FCM)was used to detect the effects of different concentrations of FC-007(0,10,20μmol/L)on the cell cycle and apoptosis of U87MG and U118MG cells.Western blotting was used to detect the effects of different concentrations of FC-007(0,25,50,100μmol/L)on the expression of matrix metalloproteinase(MMP)-2 and MMP-9 proteins,and real-time quantitative reverse transcription polymerase chain reaction(RT-qPCR)was used to detect the effects of different concentrations of FC-007(0,5,10,20μmol/L)on Nrf2 and its downstream genes HO-1,NQO-1 expression levels.The groups were compared by F tests.Results The results of CCK-8 assay showed that the median proliferation inhibitory concentrations(IC50)of U87MG and U118MG cells after FC-007 treatment for 48 h were 9.77 and 11.10μmol/L respectively.The FCM results showed that with the increase of FC-007 concentration,the proportion of cells in the G0/G1 phase was significantly higher than that in the blank control group,and the proportion of cells in the S phase was significantly lower than that in the blank control group.The changes in the G2/M phase were not obvious,and the differences were all statistical significance(F=121.962,125.478,129.653,120.516,129.340,122.425,P<0.05).After treatment of gloma cells with FC-007 for 24 h,the apoptosis rate under the concentrations of FC-007(0,10,20μmol/L)in U87MG cells[(6.4±0.3)%,(18.4±1.2)%,(26.1±1.7)%]and U118MG cells[(5.0±0.7)%,(28.4±5.6)%,(33.1±4.1)%]showed statistically significant difference(F=265.502,260.378,P<0.05).Western blotting results showed that FC-007 significantly reduced the expression of MMP-2 and MMP
作者
杨福伟
孙利波
陈广永
张金男
Yang Fuwei;Sun Libo;Chen Guangyong;Zhang Jinnan(Department of Neurosurgery,China-Japan Union Hospital of Jilin University,Changchun 130033,China)
出处
《中华实验外科杂志》
CAS
北大核心
2021年第9期1663-1666,共4页
Chinese Journal of Experimental Surgery
基金
吉林省科技厅自然科学基金(20190201030JC)。
关键词
胶质母细胞瘤
增殖
凋亡
侵袭
Glioblastoma
Proliferation
Apoptosis
Invasion
