MGMTsiRNA联合替莫唑胺提高耐药黑色素瘤细胞抗肿瘤疗效的研究  

Study on MGMTsiRNA combined with temozolomide enhances antitumor efficacy in drug-resistant melanoma cells

作　　者：申超南 李娜 蒋韬璇 杨春生 SHEN Chao-nan;LI Na;JIANG Tao-xuan;YANG Chun-sheng(Department of Medicine,Xuzhou Medical University,Xuzhou 221004,Jiangsu Province,China;Department of Dermatology,the Affiliated Huai'an Hospital of Xuzhou Medical University,Huai'an 223002,Jiangsu Province,China)

机构地区：[1]徐州医科大学医学系,江苏徐州221004 [2]徐州医科大学附属淮安医院皮肤科,江苏准安223002

出　　处：《中国临床药理学杂志》2025年第1期55-59,共5页The Chinese Journal of Clinical Pharmacology

基　　金：国家自然科学基金资助项目(81803151)。

摘　　要：目的探究O6-甲基鸟嘌呤-DNA甲基转移酶(MGMT)干扰联合替莫唑胺(TMZ)提高替莫唑胺对人耐药黑色素瘤细胞A375/TMZ的治疗作用。方法将A375/TMZ细胞随机分为对照组(正常培养)、MGMTsiRNA组(200 nmol·L^(-1)MGMTsiRNA)、实验组(1600μmol·L^(-1)TMZ)和联合组(转染MGMTsiRNA后加1600μmol·L^(-1)TMZ)。培养24 h后,用细胞计数试剂盒-8法分析细胞的增殖情况,用蛋白印迹法检测细胞中多聚ADP核糖聚合酶(PARP)、裂解的PARP(cleaved-PARP)、DNA依赖性蛋白激酶催化亚基(DNA-PKcs)和核因子κB(NF-κB)蛋白的表达水平,用免疫荧光法检测细胞中NF-κB蛋白的表达和分布。结果对照组、MGMTsiRNA组、实验组和联合组的细胞抑制率分别为0、(3.45±1.53)%、(51.24±2.73)%和(70.69±4.48)%,PARP蛋白相对表达水平分别为0.45±0.08、0.47±0.06、0.33±0.04和0.14±0.03,cleaved-PARP蛋白相对表达水平分别为0.01±0.02、0.01±0.01、0.18±0.03和0.36±0.04,DNA-PKcs蛋白相对表达水平分别为0.09±0.03、0.07±0.02、0.32±0.02和0.39±0.04,NF-κB蛋白相对表达水平分别是0.35±0.04、0.36±0.05、0.20±0.02和0.15±0.02。联合组的上述指标与实验组或对照组比较,在统计学上差异均有统计学意义(均P<0.05)。免疫荧光分析显示,对照组、MGMTsiRNA组、实验组和联合组的NF-κB平均荧光强度分别为(5.26±1.05)%、(7.58±1.18)%、(10.56±1.99)%和(15.47±2.61)%,联合组与对照组和MGMTsiRNA组的细胞相比,NF-κB在肿瘤细胞核内明显增多,在统计学上差异均有统计学意义(均P<0.01)。结论MGMTsiRNA联合TMZ可以进一步促进TMZ对耐药黑色素瘤A375/TMZ细胞的增殖抑制和凋亡。Objective To investigate whether O6-methylguanine-DNA methyltransferase(MGMT)interferencecombined with temozolomide(TMZ)could enhance the therapeutic effecttof temozolomide on human drug-resistant melanoma cells A375/TMZ.Methods A375/TMZ cells were randomly divided into 4 groups,control group(normal culture),MGMTsiRNA group(200 nmol·L^(-1) MCMTsiRNA),experimental group(1600μmol·L^(-1) TMZ)and combined group(transfection of MGMTsiRNA followed by addition of 1600μmol·L^(-1)TMZ).After 24 h of culture,the proliferation of cells in each group was analyzed by cell counting kit-8 method.Western blotting was used to detect the expression levels of poly ADP-ribose polymerase(PARP),cleaved PARP(cleaved-PARP),DNA-dependent protein kinase catalytic subunit(DNA-PKcs)and nuclear factor kappa-B(NF-κB)proteins in the cells.The expression and distribution of NF-κB proteins in the cells were detected by immunofluorescence.Results Cell inhibition rates of control,MGMTsiRNA,experimental and combined groups were 0,(3.45±1.53)%,(51.24±2.73)% and(70.69±4.48)%;the relative expression levels of PARP protein were 0.45±0.08,0.47±0.06,0.33±0.04,0.14±0.03;the relative expression levels of the cleaved-PARP protein were 0.01±0.02,0.01±0.01、0.18±0.03 and 0.36±0.04;the relative expression levels of DNA-PKcs protein were 0.09±0.03,0.07±0.02,0.32±0.02 and 0.39±0.04;the relative expression levels of NF-κB protein were 0.35±0.04,0.36±0.05,0.20±0.02 and 0.15±0.02.Compared with experimental group or control group,the differences of above indexes were all statistically significant(all P<0.05).Immunofluorescence analysis showed that the average fluorescence intensity of NF-κB in control group,MGMTsiRNA group,experimental group and combined group were(5.26±1.05)%,(7.58±1.18)%,(10.56±1.99)% and(15.47±2.61)%;and compared with the cells in control group and MGMTsiRNA group,combined group showed NF-κB was significantly increased in the nucleus of tumor cells,and the difference was statistically significant(all P<0.01).Concl

关 键 词：替莫唑胺 O6-甲基鸟嘌呤-DNA甲基转移酶 化疗耐药 恶性黑色素瘤 细胞凋亡 

分 类 号：R979.1[医药卫生—药品]