曲安奈德对高糖培养RPE细胞ICAM-1和ILK表达的影响

Effect of triamcinolone acetonide on expression of ICAM-1 and ILK in RPE cells cultured by high glucose

目的检测高糖诱导的视网膜色素上皮(retinal pigment epithelium,RPE)细胞间黏附分子(intercellular adhesion mole-cule-1,ICAM-1)和整合素连接激酶(integrin-linked kinase,ILK)的表达,并观察曲安奈德(triamcinolone acetonide,TA)对二者表达的影响,探讨炎症在糖尿病视网膜病变(diabetic retinopathy,DR)发病过程中的作用。方法体外培养人RPE细胞,应用免疫荧光细胞染色法、Western-blot法,分别检测在低糖对照组(5.6mmol.L-1葡萄糖的DMEM培养液)、高糖组(30.0mmol.L-1葡萄糖的DMEM培养液)、TA组(0.1mol.L-1TA+30.0mmol.L-1葡萄糖的DMEM培养液)RPE细胞ILK和ICAM-1蛋白的表达,实时荧光定量PCR检测二者mRNA的表达。结果 Western-blot和免疫荧光细胞染色都显示RPE细胞在高糖组培养6h时ICAM-1表达量最高,为0.378±0.012,是对照组0.220±0.008的1.71倍,TA对其表达有抑制作用,抑制率为26.98%(P<0.05);高糖组作用2h ILK蛋白表达量最高,是对照组的1.45倍,TA组中虽然ILK表达量较高糖组减少(为高糖组的76.20%),但差异无统计学意义(P>0.05),TA对其表达无明显抑制作用。而实时荧光定量PCR检测显示在在高糖组作用1h后RPE细胞表达ICAM-1、ILK mRNA开始上升,ICAM-1mRNA表达在2h达到高峰,2h时TA对高糖组ICAM-1mRNA的抑制率为83.67%,差异具有统计学意义(P<0.05)。TA组ILK mRNA的表达与高糖组差异无统计学意义(P>0.05)。结论 RPE细胞在高糖环境下能高表达ICAM-1和ILK,高糖对RPE细胞表达ICAM-1的上调作用能被糖皮质激素TA抑制,而ILK的上调不受其抑制。提示炎症在早期DR病理过程中有重要作用,RPE细胞可能通过这两种因子的信号途径参与DR的病变。

Objective To investigate effect of inflammation on diabetic retinopathy（DR）by detecting expression of intercellular adhesion molecule-1（ICAM-1）and integrin-linked kinase（ILK）in retinal pigment epithelial（RPE）cells induced by high glucose and observing effect of triamcinolone acetonide（TA）on their expression.Methods Human RPE cells were cultured in vitro.Immunofluorescent staining and Western-blot was used to detect expression of ILK and ICAM-1 in PRE cells of high glucose group（DMEM culture medium with 30.0 mmol·L-1 glucose）,TA group（DMEM culture medium with 0.1 mmol·L-1 and 30.0 mmol·L-1）,respectively.RT-PCR was used to detect expression of mRNA of ILK and ICAM-1.Results Western-blot and immunofluorescent staining showed high expression of RPE cells in high glucose group for culturing 6 hours.It was 0.378±0.012 and 1.71 times to 0.220±0.008 in control group.TA had inhibitive effects on its expression,which was 26.98%（P0.05）.Expression of ILK protein was the highest in high glucose group at 2 hours,was 1.45 times to that of control group,and was higher than that in TA group,which was 76.20% of high glucose group.There was no significant difference（P0.05）.TA had no obvious inhibition on expression of ILK.RT-PCR showed that expression of ICAM-1 and ILK mRAN was gradually increased in RPE cells in high glucose group at 1 hour,expression of ICAM-1 mRNA went to peak at 2 hours,inhibitive rate of TA on ICAM-1 mRNA was 83.67% at 2 hours,there was statistical difference（P0.05）.There was no difference in expression of ILK mRNA between TA group and high glucose group（P0.05）.Conclusions There are high expression of ICAM-1 and ILK in RPE cells under high glucose circumstance.Glucocorticoid TA can inhibit up-regulation of ICAM-1 in RPE cells induced by high glucose,while up-regulation of ILK is not inhibited,which indicate that inflammation plays a role in pathology of DR at early stage,and RPE cells may play a part in DR with the signaling pathway of these two factors.