糖尿病足伤口皮肤细胞凋亡情况及 AGEs对人皮肤成纤维细胞凋亡的影响

摘要

目的：检测糖尿病足伤口皮肤细胞凋亡情况，探讨晚期糖基化终产物（AGEs）对人皮肤成纤维细胞凋亡的影响。方法：选择36例足部伤口患者，糖尿病组18例，非糖尿病组18例。对2组临床和生化数据进行统计分析。采用免疫组化和TUNEL法检测伤口皮肤细胞凋亡情况。体外培养人原代皮肤成纤维细胞，分别给予正常浓度糖、持续高糖、波动高糖和AGEs干预72 h，采用蛋白质印迹法和流式细胞术测定细胞凋亡情况。结果：与非糖尿病组相比，糖尿病组血糖水平明显增高，伤口持续时间长（P＜0．05）。 Cleaved caspase-3在非糖尿病组和糖尿病组的免疫反应评分分别为1．04±0．23和3．04±0．31（P＜0．05）；TUNEL检测非糖尿病组和糖尿病组的细胞凋亡指数分别为（3．8±0．8）％和（8．4±1．5）％（ P＜0．05）。人原代皮肤成纤维细胞在正常浓度糖、持续高糖、高糖波动、AGEs干预下，cleaved caspase-3蛋白表达水平分别为0．80±0．13、1．22±0．18、1．46±0．32和1．83±0.25，凋亡率分别为（2．43±0．19）％、（2．89±0．51）％、（3．99±0．24）％和（6．83±0．36）％。 AGEs 组 cleaved caspase-3蛋白表达水平和凋亡率均明显高于正常浓度糖组和持续高糖组（P＜0．05）。结论：糖尿病皮肤创面细胞凋亡增加，可能是糖尿病皮肤伤口难愈的重要原因之一。与持续高糖、高糖波动组相比，AGEs促进人皮肤成纤维细胞凋亡的作用更强。%[ABSTRACT]AIM:Toinvestigatecellapoptosisindiabeticfootulcersandtheeffectofadvancedglycosylation end products (AGEs) on apoptosis in human fibroblast cells.METHODS: Diabetic foot patients (n=18) and 18 age-matched non-diabetic controls were recruited .The clinical and biochemical features were compared by statistics methods . Skin biopsies were obtained from foot .Cleaved caspase-3 was measured by immunohistochemistry using the technique of streptavidin-biotin complex ( SABC ) staining.Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling ( TUNEL) technique was used to detect apoptosis of the skin tissues .Human primary foreskin fibroblasts were isolated and cultured in the presence of 5.6 mmo/L glucose, 25 mmo/L glucose, fluctuant glucose ( changing the glucose from 5.6 mmo/L to 25 mmo/L every 8 h) or AGEs (150 mg/L, containing 5.6 mmo/L glucose).After 72 h treatment, Western blotting was used to determine the levels of the apoptotic protein cleaved-caspase-3.Other cells were trypsinized , washed with cold PBS and incubated with PI and Annexin V-FITC, then analyzed by flow cytometry to detect cell apoptosis .RE-SULTS:Diabetic patients had higher levels of fasting blood glucose (FBG), 2-hour postprandial blood glucose (2 h PBG) and glycosylated hemoglobin A1c (HbA1c), and longer wound duration.The protein level of cleaved caspase-3 was signifi-cantly higher in diabetic group , suggesting that apoptosis was increased in diabetic skin tissues .TUNEL analysis showed that apoptotic index was higher in diabetic group compared with that in non-diabetic group (8.4%±1.5% vs 3.8%± 0.8%) , which further confirmed that cell apoptosis was increased in diabetic foot tissues .In human fibroblasts , the levels of cleaved caspase-3 in normal group , sustained high glucose group , fluctuant high glucose group and AGEs group were 0.80 ±0.13, 1.22 ±0.18, 1.46 ±0.32 and 1.83 ±0.25, respectively.The apoptotic rates detected by flow cytometry were 2.43%±0.19%, 2.89%±0.51%, 3.99% ±0.24% and 6.83% ±0.36%, respectively.Both the level of cleaved caspase-3 and the apoptotic rate in AGEs group were higher than those in normal glucose group and sustained high glucose group .CONCLUSION:Increased apoptosis in diabetic foot ulcers is one of the most important reasons for im-paired wound healing .As compared to sustained high glucose and glucose fluctuations , AGEs induce greater apoptosis in human fibroblast cells .