高糖对人腹膜间皮细胞间粘合连接复合体的影响

Effect of high glucose on intercellular adherens junction complex in cultured human peritoneal cells in vitro

目的 :研究高糖对人腹膜间皮细胞 (HPMC)间粘合连接复合体表达的影响 ,探讨腹膜透析 (PD)中腹膜间皮层完整性损伤的机制。 方法 :采用人腹膜间皮细胞株HMrSV 5为研究对象。细胞分为 3组 :①正常对照组 (含 5 5mmol/LD 葡萄糖 ) ;②高糖组 (含 140mmol/LD 葡萄糖 ) ;③高渗组 (含 5 5mmol/LD 葡萄糖及 134 5mmol/L甘露醇 ) ,各组分别于 6、12、2 4、48h进行实验。采用半定量逆转录聚合酶链反应 (RT PCR)、Westernblot及免疫荧光等方法检测粘合连接复合体主要成分E Cadherin、β Catenin基因及蛋白水平表达 ,采用免疫荧光技术观察高糖对细胞Actin微丝骨架的影响。 结果 :①高糖致细胞脱落呈时间依赖效应 ;②高糖引起E Cadherin、β CateninmRNA及蛋白表达下降 ,免疫荧光显示二者在HPMC细胞膜上的表达明显减低 ,呈不连续分布 ;③高糖使Actin微丝骨架解体 ;④高渗透压组未见上述效应。 结论 :高糖使HPMCE Cadherin、β Catenin基因及蛋白表达下降 ,微丝骨架解体 ,从而损害细胞间粘合连接复合体的形成 ,造成腹膜间皮完整性的损害。

Objective:To investigate the effect of the high glucose concentration on the intercellular adherens junction complex in the cultured human peritoneal mesothelial cells(HPMC),and clarify the cause of decreased dialysis efficiency during prolonged peritoneal dialysis. Methodology:All test were performed on the human peritoneal mesothelial cell line(HMrSV 5).Three groups were divided.They were ①normal glucose group treated with 5.5 mmol/L D-glucose;②high glucose group treated with 140 mmol/L D-glucose;and ③high osmolality group with 5.5 mmol/L D-glucose and 140 mmol/L mannitol.Cell detachment and viability were observed at the time of 6,12,24,and 48 hours.The mRNA expression of E-Cadherin and β-Catenin was detected by semi quantitative reverse transcription polymerase chain reaction (RT-PCR),and the protein measured by Westernblot and immunofluorescence.High glucose-induced actin disassembly was determined by fluorescence labeling technique. Results:High glucose caused a time-dependent increase in the cell detachment.The mRNA expression and level of intercellular adherens junction protein(E-Cadherin and β-Catenin)were decreased,and the fluorescence expression immunostaining by anti-E-Cadherin and anti-β-Catenin antibodies was weaker and often discontinuous along the cell contour.When HPMC was incubated in high glucose,F-actin bundles appeared less prominent and changed to irregular network.Mannitol had no effect on actin assembly and on the expression of E-Cadherin and β-Catenin at the same osmolality. Conclusion:These findings suggest that the peritoneal mesothelium is damaged by prolonged peritoneal dialysis using high glucose dialysate,and that impairment of the intercellular adherens junction complex of HPMC by high glucose play an important role in cell detachment and a progressive reduction in dialysis efficiency.