节点文献
扇贝多肽经由EGFR/AKT/CyclinD1/CDK-4通路抑制UVA诱导的HaCaT细胞凋亡
【作者】 闫琳；
【导师】 王春波；
【作者基本信息】 青岛大学 ， 药理学， 2010， 硕士
【摘要】 目的建立8J·cm-2紫外线A(UVA)辐射损伤永生化的人角质形成细胞株(HaCaT)细胞的病理模型,经由信号通路的表皮生长因子受体(EGFR),磷脂酰肌醇-3激酶(AKT),细胞周期蛋白(CyclinDl)至周期蛋白依赖激酶(4 CDK-4)角度研究扇贝多肽(Polypeptide from Chlamys farreri, PCF)抑制UVA诱导HaCaT细胞凋亡的分子机制。方法采用琼脂糖凝胶电泳检测细胞凋亡；RT-PCR法检测胞内表皮生长因子受体EGFR表达；RACE法构建EGFR的全长cDNA文库,基因测序观察EGFR经UVA辐射诱导后的变化；蛋白印迹法检测AKT,p-AKT, CyclinDl及CDK-4的蛋白表达水平。结果EGFR抑制剂AG1478和AKT抑制剂PHZ1023均可阻断UVA引起的细胞凋亡,1.42-5.68 mmol.L-1范围内的PCF可剂量依赖性抑制UVA辐射后细胞内EGFR的表达量；EGFR经8 J·cm-2 UVA辐射1h后基因产生突变,而加入2.84 mmol.L-1PCF孵育2h后可部分修复缺失的碱基片段；预先加入AG1478和PHZ1023则分别抑制UVA引起的AKT及CyclinDl, CDK-4蛋白水平的表达。结论PCF可以通过阻断EGFR-CDK-4通路来抑制UVA诱导的HaCaT细胞凋亡。
【Abstract】 Objective We aim to investigate whether PCF protects HaCaT cells from apoptosis induced by 8 J.cm-2 UVA and explore related molecular mechanisms from EGFR to CDK-4. Methods The apoptosis was analyzed by agarose gel electrophoresis。A DNA fragmentation assay for genetic transformation, detection of EGFR by RT-PCR. AKT, Phosphorylated AKT, activated CyclinD1, and CDK-4 were investigated by western blotting. Results showed that AG1478 and PHZ1023, corresponding the inhibitor of EGFR and AKT could significantly prevente UVA-induced apoptosis of HaCaT cells. PCF not only strongly reduced the AKT production, but also diminished expression of phosphorylated AKT, CyclinDi and CDK-4 in HaCaT cells radiated by UVA in a dose-dependent manner. Pretreatment with AG1478, EGFR inhibitor or AKT inhibitor PHZ1023 was found to effectively prohibit UVA-induced apoptosis, and AG1478 markedly blocked phosphorylation of AKT. Conclution PCF obviously protects HaCaT cells from apoptosis induced by UVA and protective effects may attribute to decreasing intracellular EGFR level and blocking EGFR/CDK-4 apoptotic signaling pathway.