晚期糖基化终末产物对表皮角质形成细胞细胞周期的调控及信号通路的影响

目的 了解晚期糖基化终末产物(AGE)对表皮角质形成细胞细胞周期的影响及其可能的信号通路. 方法 体外制备AGE修饰的蛋白质(AGE-HSA,150 mg/L)作为干预手段.将原代培养的表皮角质形成细胞分为:正常组,采用无血清DK-SFM培养液培养;AGE干预组,采用含150mg/L AGE-HSA的DK-SFM培养液培养;对照组以10μmol/L UO126处理后同正常组条件培养;干预对照组以上述UO126处理后同AGE干预组条件培养.采用流式细胞仪检测细胞周期,蛋白质印迹法检测细胞周期蛋白D1、B1以及细胞周期素依赖性激酶4(CDK4)和p44/42丝裂原活化蛋白激酶(MAPK)的表达. 结果 与正常组比较,AGE干预组S期和G2/M期细胞百分比均显著降低(P＜0.05);对照组、干预对照组G2/M期细胞百分比亦显著降低,分别为(9.7±1.1)%、(9.8±0.7)%(P＜0.05).与正常组比较,其余3组细胞周期蛋白D1表达下降,其中干预对照组该蛋白仅有微弱表达;正常组、AGE干预组CDK4、细胞周期蛋白BI、p44/42 MAPK蛋白表达相似,对照组、干预对照组的3种蛋白表达显著低于前2组. 结论 AGE通过下调细胞周期蛋白D1的表达阻碍表皮角质形成细胞的细胞周期进程.

Effect of advanced glycosylation end products on cell cycle of epidermal keratinocyte and the role of signal pathway

OBJECTIVE: To investigate the effect of advanced glycosylation end products (AGE) on cell cycle of epidermal keratinocyte and its possible signal pathway. METHODS: 150 mg/L AGE-human serum albumin (AGE-HSA) was prepared in vitro. Primary cultured keratinocytes in logarithmic growth phase were harvested and divided randomly into: A group with treatment of defined keratinocyte-SFM (DK-SFM) serum-free medium], B group (with treatment of DK-SFM medium including 150 mg/L AGE-HSA), C group (with DK-SFM medium after treatment of U0126) and group D (with D K-SFM medium including 150 mg/L AGE-HSA after treatment of U0126). Cell cycle distributions were analyzed by flow cytometer. The protein levels of cyclin D1, cyclin B1, CDK4 and p44/42 MAPK were measured by Western blot. RESULTS: Compared with those of A group, the percentage of S-phase and G2/M-phase keratinocytes were decreased obviously in B group, the percentages of G2/M -phase keratinocytes showed the same tendency in C and D groups (9.7 +/- 1.1)% , (9.8 +/- 0.7)%, respectively, P <0.05]. Compared with that of A group, the expression of cyclin D1 were decreased significantly in other groups, among which a weak expression was showed in D group. There was no obvious difference between A and B groups in CDK4, or cyclin B1 and p44/42 MAPK protein levels ,which were significantly higher than those in C and D groups. CONCLUSION: AGEs inhibit the progress of cell cycle of keratinocytes by downregulation of cyclin D1 expression.