五味子甲素通过ATP结合盒转运子B1逆转胶质瘤干/祖细胞耐药性研究  

Study on schizandrin A reversing drug resistance of glioma stem/progenitor cells by ATP binding cassette subfamily B member 1

作　　者：魏子龙 狄冲 楼美清[3] 赵耀东[3] WEI Zi-long DI Chong LOU Mei-qing ZHAO Yao-dong(Department of Neurosurgery, Shanghai Pudong Hospital, Shanghai 201399, China Department of Intensive Care Unit, the Affiliated Hospital of Xuzhou Medical University, Xuzhou 221002, Jiangsu, China)

机构地区：[1]上海市浦东医院神经外科,201399 [2]徐州医科大学附属医院重症医学科,221002 [3]上海市第一人民医院神经外科,200080

出　　处：《中国现代神经疾病杂志》2017年第6期439-445,共7页Chinese Journal of Contemporary Neurology and Neurosurgery

基　　金：上海市卫生和计划生育委员会科研课题(项目编号:201540412);上海市浦东医院基金资助项目(项目编号:201313);上海市浦东医院2012年度优秀人才"南菁奖"项目~~

摘　　要：目的探讨五味子甲素对胶质瘤干/祖细胞耐药性的影响及作用机制。方法自人胶质瘤细胞系SHG-44中分离培养胶质瘤干/祖细胞SHG-44s,予五味子甲素0、12.50、25.00和50.00μmol/L联合长春新碱400、800和1200 nmol/L,细胞活性检测试剂盒CCK-8细胞毒性实验检测SHG-44s细胞增殖活性,罗丹明123染色检测SHG-44s细胞泵出药物能力,实时聚合酶链反应(PCR)和Western blotting法检测SHG-44s细胞ATP结合盒转运子B1(ABCB1)基因转录和翻译能力。结果五味子甲素50μmol/L即可抑制SHG-44s细胞增殖活性(P=0.001,0.001,0.039),剔除这一浓度后无论长春新碱浓度为400、800或1200 nmol/L,联合应用五味子甲素均可抑制SHG-44s细胞增殖活性(长春新碱400 nmol/L组:P=0.007,0.001;长春新碱800 nmol/L组:P=0.001,0.000;长春新碱1200 nmol/L组:P=0.000,0.000)。倒置荧光显微镜观察,五味子甲素12.50μmol/L组和25.00μmol/L组SHG-44s细胞可见明显绿色荧光。流式细胞术显示,随着五味子甲素浓度的增加,SHG-44s细胞罗丹明123染色阳性细胞比例分别为10.40%、39.20%和45.20%。实时PCR法显示,五味子甲素12.50μmol/L组和25.00μmol/L组SHG-44s细胞ABCB1基因表达水平较0μmol/L组降低(P=0.027,0.006),尤以25.00μmol/L组显著(P=0.034)。Western blotting法显示,随着五味子甲素浓度的增加,SHG-44s细胞P-糖蛋白表达水平下降。结论五味子甲素通过抑制胶质瘤干/祖细胞表面已存在的ABCB1基因编码的P-糖蛋白泵出药物能力并降低ABCB1基因转录和翻译能力,逆转胶质瘤干/祖细胞耐药性。Objective To investigate the effect of schizandrin A on drug resistance of glioma stem/ progenitor cells （GSPCs） and its mechanism. Methods Isolate and culture SHG-44s cells from human glioma cell line SHG-44. The SHG-44s cells were treated with different concentrations of schizandrin A （0, 12.50, 25.00 and 50.00 μmol/L） and vincristine （400, 800 and 1200 nmol/L）. The cell proliferative activity was measured by cell counting kit-8 （CCK-8） assay. Rhodamine 123 staining was used to detect the drug delivery ability of SHG- 44s cells. The transcription and translation ability of ATP binding cassette subfamily B member 1 （ABCB1） gene of SHG-44s cells was detected by real-time polymerase chain reaction（PCR） and Western blotting. Results The proliferative activity of SHG-44s cells was inhibited when the concentration of schizandrin A was 50 trmol/L （P = 0.001, 0.001, 0.039）, so this concentration was removed in the follow-up study. No matter the concentration of vincristine was 400, 800 or 1200 nmol/L, combining with schizandrin A could inhibit the proliferative activity of SHG-44s cells （vincristine 400 nmol/L： P = 0.007, 0.001; vincristine 800 nmol/L： P = 0.001, 0.000; vincristine 1200 nmol/L： P = 0.000, 0.000）. Inverted fluorescence microscopy findings showed SHG-44s cells in the group of schizandrin A 0 μmol/L rarely revealed green fluorescence, while SHG-44s cells in the groups of schizandrin A 12.50 and 25.00 μmol/L presented obvious green fluorescence. Flow cytometry showed that with the increasing of schizandrin A concentration, the percentage of positive cells by Rhodamine 123 staining was 10.40%, 39.20% and 45.20%, respectively. Real-time PCR showed that ABCB1 gene expression levels of SHG-44s cells in schizandrin A 12.50 μmol/L group and 25.00 ~mol/L group were significantly decreased comparing with schizandrin A 0 μmol/L group （P = 0.027, 0.006）, especially in schizandrin A 25.00 μmol/L group （P = 0.034）. Western blotting showed that the expression level of

关 键 词：五味子素 ATP结合匣式转运子 神经胶质瘤 肿瘤干细胞 药物耐受性 显微镜检查 荧光 流式细胞术 聚合酶链反应 免疫印迹法 肿瘤细胞 培养的 

分 类 号：R739.4[医药卫生—肿瘤]