| 摘要: |
| 目的研究高糖记忆对人系膜细胞(HMCs)表达局部醛固酮系统、活性氧(ROS)及癌胚纤维连接蛋白(oncofetal FN)mRNA水平的影响,并进一步探讨局部醛固酮系统在其中的作用。方法实验细胞分组如下:正糖组(NG,5 mmol/L D-葡萄糖培养2 d)、高糖组(HG,25 mmol/L D-葡萄糖培养2 d)、记忆组(M,25 mmol/L D-葡萄糖培养2 d→5 mmol/L D-葡萄糖培养4 d)、记忆+依普利酮组(MY,25 mmol/L D-葡萄糖培养2 d→5 mmol/L D-葡萄糖+10 μmol/L依普利酮培养4 d)、正糖+依普利酮组(NY,5 mmol/L D-葡萄糖培养2 d→5 mmol/L D-葡萄糖+10 μmol/L依普利酮培养4 d)、持续正糖组(SN,5 mmol/L D-葡萄糖培养6 d)。荧光显微镜及酶标仪检测细胞内ROS水平,蛋白质印迹法检测醛固酮合酶蛋白(CYP11B2)水平,RT-PCR检测11β-羟基类固醇脱氢酶Ⅱ、CYP11B2、oncofetal FN mRNA表达水平,激光共聚焦显微镜观察盐皮质激素受体(MR)表达及转位,放射免疫法测定上清液醛固酮水平。结果(1)HG组和M组诱导人系膜细胞CYP11B2 mRNA及蛋白分别为NG组的3.45、2.09倍和3.14、2.06倍,HG组和M组细胞上清液醛固酮含量分别为NG组的2.01、1.81倍(P均<0.05),HG组和M组均可促进MR蛋白发生核转位,定量分析示HG组和M组胞质/胞核荧光强度较NG组分别降低30%、21%(P均<0.05)。(2)HG组和M组oncofetal FN mRNA、ROS表达增加,分别为NG组的2.23、1.99倍和2.16、1.90倍(P均<0.05),MY组ROS、oncofetal FN mRNA表达分别较M组降低35%、51% (P均<0.05)。结论HMCs存在高糖记忆效应,局部醛固酮系统可能介导了高糖致系膜细胞损伤的记忆作用。 |
| 关键词: 局部醛固酮系统 代谢记忆 高糖 癌胚纤维连接蛋白 活性氧 |
| DOI：10.3724/SP.J.1008.2012.001166 |
| 投稿时间：2012-08-13修订日期：2012-09-24 |
| 基金项目:无 |
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| Local aldosterone system mediated “hyperglycaemic memory” in human mesangial cells |
| DU Chao1,XIONG Qin-pan2,ZHOU Bo1*,SU Hong1 |
(1. Department of Endocrinology, The First Affiliated Hospital, Chongqing Medical University, Chongqing 400016, China
2. Department of Intensive Care Unit, The people’s Hospital of Yuechi, Yuechi 638300, Sichuan, China
*Corresponding author.) |
| Abstract: |
| Objective To investigate the effect of hyperglycaemic memory on the local aldosterone system, reactive oxygen species (ROS) and expression of oncofetal fibronectin (oncofetal FN) mRNA in human mesangial cells (HMCs),and to further understand the role of local aldosterone system in the process. Methods In this study HMCs were divided into the following groups:normal glucose group (NG, 5 mmol/L D-glucose for 2 days),high glucose group (HG, 25 mmol/L D-glucose for 2 days), memory group (M, 25 mmol/L D-glucose for 2 days→5 mmol/L D-glucose for 4 days),memory+eplerenone group (MY,25 mmol/L D-glucose for 2 days→5 mmol/L D-glucose+10 μmol/L eplerenone for 4 days),normal glucose+eplerenone group (NY,5 mmol/L D-glucose for 2 days→5 mmol/L D-glucose+10 μmol/L eplerenone for 4 days),and persistent normal glucose group (SN, 5 mmol/L D-glucose for 6 days). ROS levels were tested by fluorescence microscope and fluorescence microplate reader. Aldosterone synthase (CYP11B2) protein expression was detected by Western blotting analysis. The mRNA expressions of 11β-hydroxysteroid dehydrogenase type 2,CYP11B2 and oncofetal FN were detected by RT-PCR. The expression and translocation of mineralocorticoid receptor (MR) was observed by laser scanning confocal microscope (LSCM). Aldosterone level in cell culture supernatant was detected by radioimmunoassay. Results (1)CYP11B2 mRNA and protein expression in group HG and in group M were all significantly increased, being 3.45, 2.09 and 3.14, 2.06 folds of those in group NG, respectively (all P<0.05). The aldosterone levels in HMCs culture supernatant were significantly increased in group HG and group M,being 2.01 and 1.81 folds of that in group NG, respectively (P<0.05). MR was activated and translocated from the cytosol to the nucleus in group HG and group M. Quantitative analysis showed that the ratios of cytosol/nucleus fluorescence intensity in group HG and group M were decreased by 30% and 21% compared with that in group NG, respectively (all P<0.05). (2)Oncofetal FN mRNA expression and ROS levels in group HG and group M were significantly increased,being 2.23, 1.99 and 2.16, 1.90 folds those of group NG, respectively (all P<0.05). Oncofetal FN mRNA expression and ROS levels in group MY were significantly decreased,being 35% and 51% of those in group M (all P<0.05). Conclusion HMCs have hyperglycaemic memory effect, which might be mediated by the local aldosterone system. |
| Key words: local aldosterone system metabolic memory high glucose oncofetal fibronectin reactive oxygen species |
