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50.00 μmol/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50 μmol/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12.50 μmol/L 组和25.00 μmol/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12.50 μmol/L 组和25.00 μmol/L 组SHG-44s 细胞ABCB1 基因表达水平较0 μmol/L 组降低（P = 0.027，0.006），尤以25.00 μmol/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。

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

Abstract:

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 μmol/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 μ mol/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 μ mol/L group and 25.00 μ mol/L group were significantly decreased comparing with schizandrin A 0 μ mol/L group (P = 0.027, 0.006), especially in schizandrin A 25.00 μ mol/L group (P =0.034). Western blotting showed that the expression level of P-glycoprotein (P-gp) in SHG-44s cells was gradually decreased with the increasing of schizandrin A concentration.  Conclusions  Schizandrin A can inhibit the drug delivery ability of P-gp coded by ABCB1 gene existing in the surface of GSPCs. It can further reverse the drug resistance of GSPCs by reducing the transcription and translation of ABCB1 gene.

Key words: Schizandrin, ATP - binding cassette transporters, Glioma, Neoplastic stem cells, Drug tolerance, Microscopy, fluorescence, Flow cytometry, Polymerase chain reaction, Immunoblotting, Tumor cells, cultured