茜草对人神经胶质瘤U87细胞的生长及丙酮酸代谢的影响.DOC

茜草对人神经胶质瘤U87细胞的生长及丙酮酸代谢的影响（上海中医药大学，上海 201203） 摘要目的考察茜草对人神经胶质瘤U87细胞生长的影响，从及丙酮酸代谢角度探讨作用。方法体外培养U87细胞，以MTT法考察茜草不同提取物对U87细胞的生长抑制作用；人全基因组表达谱芯片初步筛选茜草醇提物抑制U87细胞的可能机制；逆转录PCR验证部分差异表达基因；碘化丙啶PI）单染法检测细胞周期分布；分光光度法检测细胞内H2O2含量、丙酮酸含量、乳酸含量、Caspase-3活力、丙酮酸激酶活力、LDH）活力、MDH）活力、SDH）活力；Western blot检测SOD1）、atalase）、、、MSH6）、PCNA）、MCT1）的蛋白表达结果以不同剂量的茜草醇提物、水提物（终浓度为200，100，50，25，12.5 mg·L-1）作用于U87细胞24，48，72 h后，U87细胞生长受到明显抑制 mg·L-1作用于U87 24 h后，表达谱芯片结果揭示其作用机制与细胞周期、凋亡、氧化—还原平衡、DNA损伤及修复、单羧酸转运等有关。茜草醇提物100，50 mg·L-1处理U87 24 h进行后续实验，逆转录PCR验证MSH6、Catalase、BAX、硫氧还蛋白相互作用蛋白（TXNIP）、PCNA mRNA表达与芯片结果一致，而DNA复制解旋酶/核酸酶2（DNA2） mRNA表达仅100 mg·L-1组与芯片结果相符；茜草醇提物可使U87细胞阻滞在S期、提高Caspase-3活力；还增加了细胞内H2O2含量、MSH6、PCNA结论茜草能有效抑制人神经胶质瘤U87细胞在体外的生长活力，作用呈现一定剂量依赖和时间依赖；提取方法以乙醇提取较好。其作用机制涉及多方面，与细胞周期阻滞、Caspase-3活化、影响氧化—还原平衡、DNA损伤及修复、细胞内丙酮酸代谢有关是一味具有潜力的抗中药。 关键词：茜草；人神经胶质瘤抗肿瘤；细胞周期；氧化还原；丙酮酸代谢；DNA损伤 Effects of Rubia cordifolia L. on Cell Growth and Pyruvate Metabolism of Human Glioblastoma U87 Cell Line LI Xiaoyun, BIAN Ka (Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China) Abstract: Objective Rubia cordifolia L. is an anticancer herb. The study is to investigate its inhibitory effects on the growth of human glioblastoma U87 cell line and to explore the underlied mechanism from the perspectives of conventional anticancer mechanism used in experimental study and pyruvate metabolism. Methods U87 cells cultured in vitro were used for the study. The effective ingredients were extracted from Rubia cordifolia L. with water (water extract) or ethanol (ethanol extract). MTT assay was applied to determine the growth inhibition. To explore the preliminary mechanism, the whole human genome oligo microarray was used. Several differentially expressed genes found by microarray were further verified by reverse transcription PCR. Cell cycle distribution was analyzed by propidium iodide (PI) staining and flow cytometry. Spectrophotometry was performed to measure the intracellular content of H2O2, pyruvate and lactate as well as a