不同剂量肝素对高糖诱导后的人腹膜间皮细胞水通道蛋白1表达的影响

Impact of different dosages' heparin on the expression of aquaporins 1 of human peritoneal mesothelial cells induced by high concentration glucose

摘要:

目的  研究不同剂量肝素对高糖诱导后的人腹膜间皮细胞水通道蛋白1（AQP1）mRNA和蛋白表达水平的影响。方法  分离并培养人腹膜间皮细胞，并用高糖（2.5%葡萄糖溶液）诱导4、8、12和24 h；采用逆转录-聚合酶链反应（RT-PCR）检测AQP1 mRNA的表达水平，从而确定表达量达峰值的诱导时间。将不同剂量的肝素添加至人腹膜间皮细胞培养液中，并经2.5%高糖溶液诱导后，用RT-PCR及免疫组织化学法检测实验组与对照组AQP1 mRNA和蛋白表达水平。结果  与对照组比较，实验组AQP1 mRNA和蛋白表达明显增加，且mRNA表达呈时间依赖性，且在高糖诱导24 h时表达量达峰值，差异有统计学意义（P <0.05）；RT-PCR与免疫组织化学结果具有一致性，均提示AQP1 mRNA和蛋白表达水平在2.5%葡萄糖+1.5×10-2 mg/ml肝素组呈现明显上调，差异有统计学意义（P <0.05）；免疫组织化学染色提示2.5%葡萄糖+1.5×10-2 mg/ml肝素组细胞可见胞核及胞浆有空泡样改变。结论  肝素在高糖环境中诱导24 h后可显著增加人腹膜间皮细胞水通道蛋白AQP1 mRNA和蛋白的表达，且大剂量的肝素能使胞浆及胞核出现明显的空泡样改变。

关键词:

肝素;人腹膜间皮细胞;水通道蛋白1

Abstract:

Objective  To investigate the impact of different dosages' heparin on the expression of aquaporin-1 (AQP1) of human peritoneal mesothelial cells (HPMCs) induced by high concentration glucose (2.5 % W/W).  Methods HPMCs were separated from patients and sub-cultured, and induced by high concentration glucose for 4, 8, 12, and 24 h respectively. This is to determinate the time when the mRNA of AQP1 reaches the maximum level. Heparin of different dosages and 2.5% glucose were added into the cell culture medium. The expression levels of AQP1 were detected by using RT-PCR and immunohistochemistry in both control and experimental groups.  Results Compared with the control group, the mRNA expression level of AQP1 was up-regulated in an time-dependant manner, with the maximum expression at 24 h induced by high concentration glucose. The protein expression level of AQP1 was also up-regulated in all experimental groups, particularly by 2.5 % glucose and 1.5 × 10-2 mg/ml heparin. The difference was of statistical significance (P < 0.05). The result of RT-PCR is consistent with that of immunohistochemistry. By using immunohistochemistry, vacuolation was observed in the nucleus and cytoplasm treated by 2.5 % glucose and 1.5 × 10-2 mg/ml heparin.  Conclusion The mRNA and protein expression of AQP1 can be up-regulated by 2.5 % glucose and heparin. Vacuolation can be observed in the nucleus and cytoplasm treated by glucose and heparin of high concentration.

Key words:

heparin; human peritoneal mesothelial cells; aquaporin 1