吴琪 陈运中 吴高明

摘 要 目的：研究丹皮酚与顺铂联用对人骨肉瘤细胞MG-63增殖、凋亡的影响及可能的作用机制。方法：取对数生长期MG-63细胞，分为空白对照组、顺铂组（4 ?mol/L）、丹皮酚组（50 mg/L）和低、中、高浓度联用组（50、100、200 mg/L丹皮酚+4 ?mol/L顺铂）。采用CCK-8法检测经药物作用24、48、72 h时的细胞增殖率，采用膜联蛋白Ⅴ-异硫氰酸荧光素/碘化丙啶双染法检测经药物作用24 h时的细胞凋亡率;采用实时荧光定量聚合酶链式反应法检测经药物作用24 h时细胞中PI3KCA、Akt、mTOR、P-gp、PTEN mRNA的相对表达量。结果：与空白对照组比较，各药物组细胞各时间点的细胞增殖率和细胞中PI3KCA、Akt、mTOR mRNA的相对表达量均显著降低，细胞凋亡率和细胞中P-gp、PTEN mRNA的相对表达量均显著升高（P<0.05或P<0.01）。与顺铂组和丹皮酚组比较，高浓度联用组细胞各时间点的细胞增殖率和细胞中PI3KCA、Akt、mTOR mRNA的相对表达量均显著降低，细胞凋亡率和细胞中P-gp、PTEN mRNA表达量均显著升高（P<0.01），中、低浓度联用组上述部分指标差异均有统计学意义（P<0.05或P<0.01）。结论：丹皮酚与顺铂联用可抑制MG-63细胞增殖，促进其凋亡;这种作用可能与下调PI3KCA、Akt、mTOR mRNA表達，上调P-gp、PTEN mRNA表达有关。

关键词 丹皮酚;顺铂;骨肉瘤;PI3K/Akt/mTOR信号通路;增殖;凋亡

ABSTRACT   OBJECTIVE：To study the effects of paeonol combined with cisplatin on the proliferation and apoptosis of human osteosarcoma cell MG-63 and its possible mechanism. METHODS： MG-63 cells in logarithmic growth phase were divided into blank control group， cisplatin group （4 ?mol/L），paeonol group （50 mg/L），and low，medium，high concentration combined groups （50，100，200 mg/L paeonol+4 ?mol/L cisplatin）. CCK-8 method was used to detect the cell proliferation rate at 24，48，and 72 hours of treatment. Annexin Ⅴ-FITC/PI double staining method was used to detect the cell apoptosis rate at 24 hours of treatment. The relative expression of PI3KCA，Akt，mTOR，P-gp and PTEN mRNA in cells were detected by qRT-PCR. RESULTS： Compared with blank control group，the cell proliferation rate at each time point， and the relative expression of PI3KCA，Akt and mTOR mRNA in cells were significantly reduced and the apoptosis rate and the relative expression of P-gp and PTEN mRNA in the cells were increased significantly （P<0.05 or P<0.01）. Compared with cisplatin group and paeonol group， cell proliferation rate at each time point and the relative expression of PI3KCA，Akt and mTOR mRNA in cells were decreased significantly in the high concentration combination group， while the relative expression of P-gp and PTEN mRNA in the cells were significantly increased （P<0.01）; there were statistical significance in some of the above indicators in the medium and low concentration combination groups （P<0.05 or P<0.01）. CONCLUSIONS：The combination of paeonol and cisplatin could inhibit the proliferation of MG-63 cells and promote their apoptosis， which may be related to the down-regulation of PI3KCA，Akt and mTOR mRNA expression，and the up-regulation of P-gp and PTEN mRNA expression.

KEYWORDS   Paeonol; Cisplatin; Osteosarcoma; PI3K/Akt/mTOR signaling pathway; Proliferation; Apoptosis

骨肉瘤是起源于间叶组织，以能产生骨样组织的梭形基质细胞为特征的骨骼系统原发性恶性肿瘤[1]，好发于骨生长活跃的青少年[2]。骨肉瘤恶性程度高、破坏性强，早期便可发生侵袭与转移[3]。随着保肢手术、放疗和新辅助化疗的发展，骨肉瘤患者的5年生存率已提高到70%左右[4]，但复发患者的生存率仍不足30%[5]。目前，临床治疗骨肉瘤的化疗药物以阿霉素、顺铂和大剂量甲氨蝶呤为主，然而高剂量化疗药物所致的毒副作用和肿瘤细胞原发或继发的多药耐药严重影响了其临床疗效和患者的长期生存率[6]。顺铂作为骨肉瘤化疗的常用药物，不仅可以通过细胞毒作用直接杀伤肿瘤细胞，还可以通过诱导细胞凋亡发挥治疗作用[7-8]，但是其肾毒性和致严重呕吐的副作用限制了该药的临床使用[9]。……