糖基化终末产物对人表皮角质形成细胞周期调控的影响机制  

Mechanisms of disordered cell cycle in keratinocytes mediated by advanced glycation end products

作　　者：董叫云[1] 牛轶雯[1] 谢挺[1] 青春[1] 陆树良[1] 

机构地区：[1]上海交通大学医学院附属瑞金医院上海市烧伤研究所,上海200025

出　　处：《创伤外科杂志》2009年第5期402-406,共5页Journal of Traumatic Surgery

基　　金：国家重点研究发展规划项目资助(G1999054205);国家自然科学基金项目(30600645)

摘　　要：目的通过体外试验探讨糖基化终末产物(AGEs)对表皮角质形成细胞周期调控的影响机制。方法体外培养的人表皮角质形成细胞经AGEs(150mg/ml)作用后,用噻唑兰(MTT)法研究其增殖活力、流式细胞仪研究细胞周期和凋亡率。用小片断RNA干扰(siRNA)的方法阻断细胞中AGEs受体(receptor of AGEs,RAGE)mRNA的表达,realtime PCR鉴定其阻断效率。RT-PCR法检测RAGE阻断前后各组细胞内RAF-1、黏着斑激酶(FAK)、周期素(cyclin)D1、cyclinB1、周期表依赖性激酶4(CDK4)的核酸水平表达差异。结果经AGEs干预后表皮角质形成细胞增殖活性下降,凋亡率升高,细胞周期G2期的比例显著增高,而细胞周期调控相关因子及信号因子RAF-1、cyclinD1、cyclinB1、CDK4的核酸表达较对照组却明显升高,用RAGE阻断以上因子后表达则与对照组相比无差异。结论在上述体外模型下,表皮角质形成细胞的周期调控因子升高,而增殖却下降且凋亡增加,可能是AGEs干预后细胞内启动内源性代偿调控机制,且这种调控机制与其受体(RAGE)途径相关;AGEs也可能还通过以上细胞周期调控因子以外的其他途径或机制影响细胞生物学功能。Objective To explore the mechanisms of disordered cell cycle in keratinocytes that were mediated by advanced glycation end pruduets （AGEs） in vitro. Methods Human epidermal keratinocytes（HEK）were incubated with or without AGEs （ 150mg/ml ）, and 150mg/ml BSA （ bovine serum albumin ）was supported in the normal medium as controls in this study. After 48 hours of incubation, cell apoptosis and cell cycle were detected through flow cytometer,and the cell growth activity was assayed by MTT method. The receptors of AGEsl RAGE） were interrupted by siRNA, and the silencing efficiency was affirmed with real-time PCR. The expressions of RAF- 1, FAK,cyclinDl ,eyclinB1, CDK4 on mRNA levels were examined with RT-PCR before and after gene silencing. Results The reduced cell proliferation and increased apoptosis were found by AGEs （ 150mg/ml） interruption. The percentages of G2 stages in cell cycle were augmented significantly and the expressions of cell cycle relative factors,such as RAF-1 ,cyclinD1 ,cyclinB1, CDK4 ,were raised significantly on mRNA levels mediated by AGEs before genes silencing. But there were no statistical differences in these factors between AGEs group and control ones after RAGE were interrupted by siRNA. Conclusion In this study model,the phenomena of increased cell cycle relative factors and inhibited cell proliferation might be one of the endogenous compensating mechanisms in keratinocytes treated with AGEs. This mechanism was correlated with RAGE signal pathway. AGEs may have impact on cell cycle in other pathways except above relative factors in the study.

关 键 词：糖基化终末产物 周期素D1 周期素B1 周期素依赖性激酶4 细胞增殖 

分 类 号：R392[医药卫生—免疫学]