高糖状态对胰腺癌细胞氧化应激水平的促进作用
High glucose promotes oxidative stress in pancreatic cancer cells

摘要：目的 研究高浓度葡萄糖对胰腺癌细胞株BxPC-3活性氧(ROS)及抗氧化酶水平的影响。方法 用DCFH-DA荧光探针检测细胞ROS水平；用过氧化氢(H2O2)试剂盒检测细胞内H2O2水平；用不同抗氧化酶试剂盒检测细胞中总超氧化物歧化酶(T-SOD)、过氧化氢酶(CAT)及谷胱甘肽过氧化物酶(GPX)活性；用Western blot方法检测细胞中SOD1和SOD2蛋白水平表达；通过siRNA技术下调SOD2的表达，研究H2O2的生成与SOD的关系。结果 胰腺癌细胞在不同浓度葡萄糖培养基中培养后，细胞内ROS水平及H2O2水平明显升高，并呈现浓度依赖性(P<0.05),甘露醇对氧化应激水平无明显影响；高糖状态可以明显提高细胞中T-SOD及CAT活性(P<0.05)，而对GPX活性无明显影响；与正常浓度葡萄糖组相比，高浓度葡萄糖可明显升高细胞SOD2蛋白表达水平，而对SOD1蛋白表达无明显影响；siRNA组SOD2蛋白表达水平显著下降(P<0.05)，下调SOD2表达可明显降低高糖状态所调控的H2O2水平。结论 高浓度葡萄糖可明显提高胰腺癌细胞氧化应激水平，H2O2的产生与SOD2的升高存在一定联系。
ABSTRACT: Objective To investigate the effects of high glucose on the levels of reactive oxygen species (ROS) and antioxidant enzymes in pancreatic cancer cell line BxPC-3. Methods After BxPC-3 cells were cultured in different concentrations of glucose, their intracellular ROS level determined using 2,7-dichlorodihydrofluorecein diacetate. The level of intracellular hydrogen peroxide (H2O2) was measured using H2O2 assay kit. The activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX) were detected by different antioxidant enzyme analysis kits. The protein levels of SOD1 and SOD2 were measured by Western blot. The effect of small interfering RNA (siRNA) knockdown of SOD2 on the level of H2O2 was also tested in pancreatic cancer cells. Results The production of ROS and H2O2 was increased by glucose in a concentration-dependent manner (P<0.05). Addition of mannitol, an osmosis regulator, did not affect the levels of ROS and H2O2. Hyperglycemia could increase the activities of T-SOD and CAT as well as the protein expression of SOD2 (P<0.05). SOD2 siRNA was successfully transfected into BxPC-3 cells, resulting in the inhibition of SOD2 protein expression (P<0.05). Down-regulation of SOD2 significantly decreased hyperglycemia-induced H2O2 level. Conclusion Hyperglycemia can increase ROS level in pancreatic cancer cells. The production of H2O2 is related to SOD2 level