【作者】 李金莲；

【导师】 王春波；

【作者基本信息】 青岛大学 ， 药理学， 2007， 硕士

【摘要】 目的建立UVA诱导的HaCaT细胞凋亡模型，从活性氧（ROS）、p38丝裂原活化蛋白激酶（p38MAPK）通路和半胱天冬酶-3（caspase-3）的角度，研究扇贝多肽（Polypeptide from Chlamys farreri，PCF）抑制UVA引起的HaCaT细胞凋亡的分子机制。方法采用正交实验设计，以流式细胞仪PI染色法测定细胞凋亡率，确立UVA诱导的HaCaT细胞凋亡模型。实验设计分为6组：对照组、UVA模型组、UVA+5.68 mmol·L^-1维生素C阳性对照组、UVA+5.69 mmol·L^-1 PCF组、UVA+2.84mmol·L^-1PCF组、UVA+1.42 mmol·L^-1PCF组。以2，7-二氯氢化荧光素二酯（DCFH-DA）为荧光探针，检测PCF对UVA照射后细胞内ROS生成的影响；琼脂糖凝胶电泳分析PCF、p38MAPK抑制剂（SB203580）及caspase-3特异性抑制剂（Ac-DEVD-CHO）对细胞凋亡的影响；Hochest 33258荧光染色观察H₂O₂引起的细胞凋亡形态学改变及PCF对凋亡的影响：蛋白质印迹法检测p38MAPK、磷酸化p38MAPK及c-fos蛋白表达；Real-Time PCR检测c-fos mRNA表达；流式细胞术检测caspase-3的活性。结果正交实验结果表明，8J·cm^-2 UVA照射HaCaT细胞后18 h为最佳凋亡模型；1.42～5.69 mmol·L^-1剂量范围内的PCF可明显抑制UVA引起的HaCaT细胞凋亡及细胞内活性氧的产生（P＜0.05）；PCF对H₂O₂引起的核固缩等细胞凋亡形态学改变有显著抑制作用；预先加入SB203580和Ac-DEVD-CHO可明显抑制UVA诱导的HaCaT细胞DNA Ladder的形成；1.42～5.69 mmol·L^-1剂量范围内的PCF可剂量依赖性抑制8J·cm^-2 UVA引起的p38MAPK磷酸化、caspase-3的活化及c-fos mRNA和蛋白的表达（P＜0.05，P＜0.01）。结论PCF可抑制8J·cm^-2UVA诱导的HaCaT细胞凋亡，其作用机理与抑制UVA诱导的ROS产生、p38MAPK通路激活和caspase-3活化有关。更多还原

【Abstract】 OBJECTIVE To establish UVA-induced apoptotic model of HaCaT cells and investigate themechanism of Polypeptide from Chlamys farreri （PCF） protecting HaCaT cells from UVA-inducedapoptosis through ROS, p38 mitogen activated protein kinase （p38MAPK） pathway and caspase-3.METHODS UVA-induced apoptotic model of HaCaT cells was established by orthogonal design andapoptotic rate was determined by flow cytometry PI staining. Cells were divided into six groups:control group, UVA model group, UVA+5.68 mmol·L^-1 vitamine C positive control group, UVA+5.69mmol·L^-1 PCF group, UVA+2.84 mmol·L^-1 PCF group, UVA+1.42 mmol·L^-1 PCF group. Effect ofPCF on UVA-induced production of ROS was detected by 2’, 7’-Dichlorofluorescin diacetate（DCFH-DA）. Using agarose gel electrophoresis, the effects of PCE p38MAPK inhibitor SB203580and caspase-3 inhibitor Ac-DEVD-CHO on UVA-induced apoptosis were investigated. Morphologyalteration caused by H₂O₂ was observed by Hoechst 33258 fluorescent staing. Protein expressionlevels of p38MAPK, phosphorylated p38MAPK and c-fos were determined by Western blot analysts.Expression of c-fos mRNA was examined by Real-Time PCR. Caspase-3 activity was assayed byflow cytometry. RESULTS Results of orthogonal experiment suggest that 8J·cm^-2 UVA and 18 hoursincubation after radiation make up the best apoptotic model. 1.42～5.69 mmol.L^-1 PCF significantlyinhibited UVA-induced apoptosis and ROS accumulation （P＜0.05）. PCF attenuated apoptoticmorphology alteration caused by H₂O₂. SB203580 and Ac-DEVD-CHO had inhibitory effects onUVA-induced apoptosis of HaCaT cells. 1.42～5.69 mmol·L^-1 PCF dose-dependently attenuatedUVA-induced p38MAPK activation, caspase-3 activation and expression of c-fos （P＜0.05, P＜0.01）.CONCLUSION PCF could protect HaCaT cells from UVA-induced apoptosis. Its inhibitory effect onapoptosis may attributes to inhibition of production of ROS, activation of p38MAPK pathway andcaspase-3.更多还原