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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 investigate the mechanism of the effect of AGEs on the cycle regulation of epidermal keratinocytes in vitro. Methods Human epidermal keratinocytes cultured in vitro were treated with AGEs (150 mg / ml), and their proliferative activity was investigated by MTT assay. The cell cycle and apoptosis rate were studied by flow cytometry. A small fragment of RNA interference (siRNA) was used to block the expression of AGEs receptor (RAGE) mRNA in cells, and its blocking efficiency was identified by realtime PCR. The expression of RAF-1, FAK, cyclin D1, cyclinB1 and CDK4 in the cells before and after RAGE block were detected by RT-PCR. Results After treated with AGEs, the proliferation of keratinocytes decreased, the apoptosis rate increased, and the proportion of cells in G2 phase increased significantly. However, the expressions of cell cycle regulatory factors and RAF-1, cyclinD1, cyclinB1 and CDK4 were significantly higher The control group was significantly increased, with RAGE blocking the above factor expression was no difference compared with the control group. Conclusions Under the above in vitro model, the epidermal keratinocyte cycle regulators are increased, but the proliferation is decreased and the apoptosis is increased, which may be due to the initiation of endogenous compensatory regulation by intracellular AGEs after intervention by AGEs (RAGE) pathway; AGEs may also affect cell biological functions through other pathways or mechanisms than the above cell cycle regulators.