晚期糖基化终末产物修饰人血清白蛋白对离体角质形成细胞迁移功能的影响

Impact of advanced glycosylation end products-modified human serum albnmin on migration of epidermal keratinocytes： an in vitro experiment

目的探讨晚期糖基化终末产物（AGE）对大鼠离体表皮角质形成细胞迁移功能的影响及其可能机制。方法制备AGE修饰人血清白蛋白（AGE—HSA），加入分离自正常SD大鼠背部表皮组织的角质形成细胞培养体系（终浓度60μg／ml，AGE—HSA组），并设空白对照组。以噻唑盐（MTT）比色法测定2组角质形成细胞培养12、24h贴壁率（以吸光度值表示）；划痕实验和Transwell实验观察细胞迁移数；流式细胞仪观察细胞整合素α3的表达；扫描电镜观察细胞伪足形成情况；免疫荧光染色法观察细胞微丝形态变化。结果AGE-HSA组和空白对照组培养12h的贴壁率分别为0．112±0．022、0．122±0．004（P〈0．05），培养24h的贴壁率分别为0．173±0．012、0．267±0．024（P〈0．05）；划痕实验迁移细胞数分别为（7±4）和（61±11）个／HP（P〈0．05），Transwell实验迁移细胞数分别为（72±18）和（288±52）个（P〈0．05）；角质形成细胞整合素α3表达量分别为（3．2±1．2）％和（36．6±11．2）％（P〈0．05）。AGE-HSA组细胞的伸展、伪足形成及微丝形成均受到抑制。结论高糖环境下AGE的大量蓄积对角质形成细胞的迁移有明显抑制作用，其作用机制可能与AGE及其受体途径以及整合素细胞内转导途径异常有关。

Objective To explore the impact of advanced glycosylation end products （ AGE）- modified human serum albumin （AGE-HSA） on keratinocte migration and the mechanism thereof. Methods AGE-HSA was prepared in vitro. Epidermal keratinocytes from Sprague-Dawley rats＇ back were cultured and treated with AGE-HSA of the terminal concentrations of 0, 30, 60, 90, 120, and 150 μg/ml for 1, 3, 5, and 7 days respectively. MTT method was used to detect the keratinocyte adhesion rate, expressed by absorbance. Keratinocyte migration ability was assessed by scratch wound healing assay and Transwell assay. Expression of intogrin α3 was determined by flow cytometry. Scanning electron and inverted microscopes were used to observe the pseudopedium and mierofilament of the keratinocytes. Immunofluorescenee staining was used to detect the form of F-actin in the cells. Results The adhesion rates of the keratinocyte cultured with AGE-HSA for 12 and 24 hours were （0. 112 ± 0. 022） and （0. 173± 0. 012 ） respectively, both significantly lower than those of the control group [ （0. 122 ± 0. 004） and （0. 267 ± 0. 024） respectively, both P〈0.05） ]. Scratch wound healing assay showed that the amount of migrating cells in the AGE-HSA group was （7 ±4）/HP, significantly less than that of the control group [ （61± 11 ）/HP, P 〈0.05） ], and Transwell assay showed that the amount of migrating cells in the AGE-HSA group was （72 ± 18）/HP, significantly less than that of the control group [ （288 ± 52 ）/HP, P 〈 0. 05 ]. The expression rate of keratinocyte integrin α3 in the AGE-HSA group was （3.2 ± 1.2）%, significantly lower than that in the control group [（36. 6 ± 11. 2）%, P 〈 0. 05]. The spreading of cell body, and the formation of pseudopodium and microfilament of the AGE-HSA group were all depressed in comparison with the control group. Conclusion Keratinocyte migration is inhibited by AGE accumulation in high glucose condition. The mechanism may be the abnormality in the integrin inside-out signaling pathway and AGE-RAGE signaling pathway.