背景与目的:吉西他滨(gemcitabine,GEM)作为治疗胰腺癌的一线化疗药物,随着其临床耐药性的出现,化疗疗效显著降低,而分泌型聚集素(secretory clusterin,sCLU)的表达与多种肿瘤细胞耐药密切相关.研究sCLU干预GEM致MIA PaCa-2氧化应激指标的改变,以期探讨GEM耐药机制.方法 :将MIA PaCa-2细胞暴露于质量浓度分别为0、0.63、1.25、2.50、5.00和10.00 μg/mL 的GEM培养液和sCLU(5.4 μmol/L)干预的0.63、1.25、2.50、5.00和10.0 μg/mL培养液中作用24 h,测定细胞增殖抑制情况,计算细胞的半数抑制浓度(50％ inhibitory concentration,IC50);将MIA PaCa-2细胞暴露于IC50的GEM培养液和sCLU干预培养6、12、 24、48和72 h,计算并观察细胞抑制率随时间变化趋势;将MIA PaCa-2细胞置于质量浓度分别为0、1.25、 2.50、5.00 μg/mL的GEM培养液和sCLU(5.4 μmol/L)干预培养24 h,测定细胞内活性氧(reactive oxygen species, ROS)表达水平及超氧化物歧化酶(superoxide dismutase,SOD)和过氧化氢酶(catalase,CAT)活力变化.结果 :细胞抑制率随GEM浓度的升高而升高,且具有明显的剂量-反应关系(P＜0.05);细胞抑制率在低质量浓度 (0.63 μg/mL)时,sCLU干预组高于GEM组,随着处理浓度增加,GEM组抑制率高于sCLU干预组(P＜0.05);GEM致MIA PaCa-2细胞IC50为2.50 μg/mL.与对照组比较,GEM、sCLU干预组ROS表达水平和SOD、CAT活力水平均升高(P＜0.05),且随着浓度的升高,ROS呈现明显剂量-反应关系,SOD、CAT呈先升高后降低趋势;相同剂量不同组间比较,sCLU干预组的ROS表达水平小于同剂量GEM药物组(P＜0.05);在GEM质量浓度为2.50和 5.00 μg/mL时,sCLU干预组SOD活力水平小于同剂量GEM药物组(P＜0.05),CAT活力水平大于同剂量GEM药物组(P＜0.05).结论 :GEM可抑制MIA PaCa-2细胞增殖,在一定药物浓度范围内,sCLU可干预GEM引起MIA PaCa-2细胞内ROS表达水平和SOD、CAT酶活力表达水平的明显变化,sCLU在一定程度可能通过调节GEM氧化损伤而产生耐药.%Background and purpose: Gemcitabine (GEM) is a first-line chemotherapy drug for pancreatic cancer. With the emergence of clinical drug resistance, the efficacy of chemotherapy has been greatly reduced, while the expression of secretory clusterin (sCLU) was closely related to chemotherapy resistance in multiple tumors. This study aimed to explore the effects of secretory clusterin on oxidative damage in MIA PaCa-2 cells treated by GEM and preliminary mechanism of resistance to GEM. Methods: MIA PaCa-2 was exposed to GEM and sCLU intervened groups with different concentrations (0, 0.63, 1.25, 2.50, 5.00 and 10.0 μg/mL) for 24 hours. The intervened concentration of GEM was 5.4 μmol/L. The inhibition rates of cell proliferation were determined by CCK-8. Cell reactive oxygen species (ROS) was measured by dichloro-dihydro-fluorescein diacetate (DCFH-DA) method. Superoxide dismutase (SOD) activity and catalase (CAT) activity were measured by their corresponding assay kits respectively. Results: Compared with the negative control group (0 μg/mL), the inhibition rates of the GEM groups and sCLU intervened groups were significantly increased (P＜0.05) in a distinct dose-effect manner. At a low concentration of 0.63 μg/mL, the inhibition rates of the GEM groups were higher than those of the sCLU intervened groups, while the trend was reversed in high concentration range. Compared with the negative control group (0 μg/mL), the intracellular ROS levels, SOD and CAT activity of the GEM and sCLU intervened groups significantly increased (P＜0.05). ROS levels presented a distinct dose-effect relationship while the SOD and CAT activities increased first and then decreased along with the increase of GEM concentrations. The ROS levels of the GEM group were lower than those of the sCLU intervened group at the same dose (P＜0.05). The SOD activities of the GEM group were higher than those of the sCLU intervened group, while the CAT activities were opposite at the concentrations of 5.00 and 10.00 μg/mL (P＜0.05). Conclusion: GEM exposure can inhibit the growth of MIA PaCa-2 cells. After GEM exposure, the ROS levels, SOD and CAT activity of MIA PaCa-2 cells can be changed by sCLU intervention. GEM resistance could be regulated by sCLU through oxidative damage effect.
展开▼
