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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(n MGMT)基因表观遗传修饰的影响。n 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 100 μmol/L、TMZ 200 μmol/L、LEN 100 μmol/L+TMZ 200 μmol/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n MGMT mRNA水平，采用甲基化特异性PCR检测细胞中n MGMT基因启动子区甲基化情况。n 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(n 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(n 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(n 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n MGMT基因启动子区均为未甲基化状态。n 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n MGMT基因启动子区甲基化状态改变无关。n “,”Objective:To investigate the effect of lenalidomide (LEN) combined with temozolomide (TMZ) on proliferation, invasion, drug resistance and O6-methylguanine-DNA methyltransferase (n MGMT) gene epigenetic modification of TMZ-resistant human glioblastoma cell line U251/TR.n 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 100 μmol/L LEN, 200 μmol/L TMZ, 100 μmol/L LEN+200 μmol/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 n MGMT mRNA expression in cells of each group was detected by reverse transcription-PCR; methylation specific PCR was used to detect the n MGMT gene promoter methylation in each group of cells.n 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 (n 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 (n 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 (n 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 and DMSO group, and the MGMT fluorescence intensity in TMZ group and LEN+TMZ group (+) was obviously lower than that in LEN group (+++) and DMSO group (+++). The MGMT gene promoter was unmethylated in all groups.n Conclusion:LEN alone does not obviously inhibit the proliferation and invasion of U251/TR cells; but LEN combined with TMZ could inhibit the proliferation and invasion of U251/TR cells and co-reverse the drug resistance of U251/TR cells, whose mechanism is not related to the changes of n MGMT gene promoter methylation.n