来那度胺联合替莫唑胺对U251/TR细胞耐药性及MGMT基因表观遗传修饰的影响  

Effect of lenalidomide combined with temozolomide on drug resistance and O6-methylguanine-DNA methyltransferase gene epigenetic modification of U251/TR cells

作　　者：潘强 朱琳 岳晓 高勇 黄华 姜宗飞 马云峰 宋纯玉 Pan Qiang;Zhu Lin;Yue Xiao;Gao Yong;Huang Hua;Jiang Zongfei;Ma Yunfeng;Song Chunyu(Department of Neurosurgery,Ji'nan People's Hospital Affiliated to Shandong First Medical University,Ji'nan Key Laboratory Neuro-oncology Molecular Biology,Ji'nan 271199,China;Department of Emergency,Ji'nan People's Hospital Affiliated to Shandong First Medical University,Ji'nan 271199,China;Department of Neurosurgery,General Hospital of Shenzhen University,Shenzhen 518055,China;Department of Cardiology,Ji'nan People's Hospital Affiliated to Shandong First Medical University,Ji'nan 271199,China)

机构地区：[1]山东第一医科大学附属济南人民医院神经外科,济南市神经肿瘤分子生物学重点实验室,271199 [2]山东第一医科大学附属济南人民医院急诊科,271199 [3]深圳大学总医院神经外科,深圳518055 [4]山东第一医科大学附属济南人民医院心内科,271199

出　　处：《中华神经医学杂志》2021年第9期865-872,共8页Chinese Journal of Neuromedicine

基　　金：国家自然科学基金(81772290);济南市卫生健康人才优秀青年技术骨干项目(济卫科教发[2019]7号);济南市卫生健康委员会科技计划项目(2020-4-126)。

摘　　要：目的探讨来那度胺(LEN)联合替莫唑胺(TMZ)对TMZ耐药性人脑胶质母细胞瘤系U251/TR细胞增殖、侵袭、耐药性及O6-甲基鸟嘌呤-DNA甲基转移酶(MGMT)基因表观遗传修饰的影响。方法将前期应用分步诱导法成功构建的U251/TR细胞分为二甲基亚砜(DMSO)组、LEN组、TMZ组及LEN+TMZ组,其中DMSO组不进行任何药物干预,LEN组、TMZ组、LEN+TMZ组分别按LEN 100μmol/L、TMZ 200μmol/L、LEN 100μmol/L+TMZ 200μmol/L浓度处理U251/TR细胞(给药1次/24 h)。处理后24、48、72、96 h时,采用磺酰罗丹明B比色分析法检测细胞增殖率;处理后96 h时,采用Transwell实验检测细胞侵袭能力,采用流式细胞术检测细胞增殖周期,采用Western blotting实验、免疫组化染色、免疫荧光染色检测细胞中MGMT水平,采用RT-PCR法检测细胞中MGMT mRNA水平,采用甲基化特异性PCR检测细胞中MGMT基因启动子区甲基化情况。结果处理后24、48、72、96 h时,LEN+TMZ组的细胞增殖率较TMZ组、LEN组、DMSO组均明显降低,差异均有统计学意义(P<0.05)。处理后96 h时,LEN+TMZ组的穿膜细胞数较其他3组均明显减少,G0/G1期细胞比例较其他3组均明显升高,差异均有统计学意义(P<0.05);TMZ组及LEN+TMZ组细胞中MGMT蛋白、mRNA水平较LEN组、DMSO组均明显降低,差异均有统计学意义(P<0.05);TMZ组及LEN+TMZ组中胞浆内MGMT表达呈强阳性或中等阳性的细胞数量较LEN组、DMSO组明显更少,MGMT荧光强度(+)较LEN组(+++)、DMSO组(+++)明显减弱。各组细胞中MGMT基因启动子区均为未甲基化状态。结论LEN单药对U251/TR细胞的增殖、侵袭无明显抑制作用,而LEN联合TMZ能抑制该细胞的增殖、侵袭并逆转其耐药性,但其机制与MGMT基因启动子区甲基化状态改变无关。Objective To investigate the effect of lenalidomide(LEN)combined with temozolomide(TMZ)on proliferation,invasion,drug resistance and O6-methylguanine-DNA methyltransferase(MGMT)gene epigenetic modification of TMZ-resistant human glioblastoma cell line U251/TR.Methods A TMZ-resistant human glioma cell line,U251/TR,was successfully established by stepwise exposure of U251 parental cells to TMZ.U251/TR cells were divided into dimethyl sulfoxide(DMSO)group,LEN group,TMZ group and LEN+TMZ group(DMSO group:without any drug intervention;LEN group,TMZ group,and LEN+TMZ group were pretreated with 100μmol/L LEN,200μmol/L TMZ,100μmol/L LEN+200μmol/L TMZ,respectively;the drugs were administered once every 24 h).The proliferation rate of these cells in each group was detected by sulfonylrhodamine B colorimetric assay at different time points(24,48,72,and 96 h after treatment).At 96 h after treatment,the invasion and migration abilities of cells in each group were detected by Transwell assay;the proliferation cycle of cells in each group was detected by flow cytometry;Western blotting,immunohistochemical staining and immunofluorescence staining were used to detect the MGMT protein expression,and the MGMT mRNA expression in cells of each group was detected by reverse transcription-PCR;methylation specific PCR was used to detect the MGMT gene promoter methylation in each group of cells.Results The cell proliferation rate of LEN+TMZ group was significantly decreased as compared with TMZ,LEN,and DMSO groups at 24,48,72 and 96 h after treatment(P<0.05).At 96 h after treatment,LEN+TMZ group had significantly decreased number of transmembrane cells,and significantly increased ratio of cells at G0/G1 phase as compared with the other 3 groups(P<0.05);the MGMT protein and mRNA expression levels in TMZ group and LEN+TMZ group were significantly lower than those in LEN group and DMSO group(P<0.05);and the number of cells with strong or moderate MGMT expression in TMZ group and LEN+TMZ group was obviously less than that in LEN group an

关 键 词：神经胶质瘤 来那度胺 替莫唑胺 耐药性 O6-甲基鸟嘌呤-DNA甲基转移酶 

分 类 号：R965[医药卫生—药理学]