空气污染PM2.5颗粒中多环芳烃诱导皮肤老化的分子机制研究

Using international standard sample of PM 2.5 polycyclic aromatic hydrocarbons(PAHs) and different culture models including human keratinocytes, fibroblasts, HaCaT cells and three dimensional artificial skin, first we plan to find the effects of PAHs on the induction of Matrix Metalloprotenases (MMPs)as well as other skin aging markers in human skin. Then we would study further the Aryl hydrocarbon receptor (AhR)on cells with the PAHs induced-skin aging model. After activating, inhibiting or knockdown the expression of AhR, a series of molecular markers both in cytoplasm and nucleus related to AhR will be detected by using RT-PCR, Western-bloting, fluorescence probe, In Situ hybridization and siRNA transfection. We hope to be able to illustrate, with the results of this part, the different cell signal transduction pathways after activating cell AhR. Finally, we are also going to explore the effects of PAHs on the telomere shortening and the transcription of gene sirtuin family using the the skin-aging model and above molecular techniques, trying to find how the polluted chemical substances like PAHs regulated the intrinsic aging in cell, and how communicated between skin extrinsic and intrinsic aging process. With all the data above, we hope to present the complex effects of PAHs on skin aging, to understand the molecular mechnism during the process, and alternatively to illustrate the hazard effects of air pollution on human skin health.

本课题采用国际标准品PM2.5芳烃化合物和人角质形成细胞、成纤维细胞以及3D人工皮肤模型等多种平台，研究多环芳烃是否可诱导基质金属蛋白酶（MMP）以及其它老化指标的高表达，初步建立空气污染诱导的细胞老化模型；然后逐层深入研究多环芳烃受体（AhR），通过特异性AhR激动剂、拮抗剂以及siRNA干扰技术分别使AhR高表达和低表达（竞争性封闭和Knockdown受体），同时检测AhR下游的胞浆内及核内一系列分子标记，借此拟阐明多环芳烃诱导皮肤老化的多种信号传导途径；最后采用原位杂交、荧光分子标记以及RT-PCR等技术研究多环芳烃对细胞染色体端粒短缩以及长寿基因sirtuin家族成员的表达影响，探索外源性化学物质对细胞内在性衰老机制的作用，从而在皮肤外源性与内源性老化之间发现内在联系。通过本课题研究，希望能完整描述多环芳烃对皮肤老化的复杂影响及其内在分子机制，阐述环境空气污染对人类皮肤的影响。

本课题采用了国际标准品PM2.5芳烃化合物和人角质形成细胞、成纤维细胞以及3D人工皮肤模型等多种平台，研究多环芳烃是否可诱导基质金属蛋白酶（MMP）以及其他老化指标的高表达，逐层深入研究多环芳烃受体（AhR），通过特异性AhR激动剂、拮抗剂以及siRNA干扰技术分别使AhR高表达和低表达，同时检测AhR下游的胞浆内及核内一系列分子标记，借此拟阐明多环芳烃诱导皮肤。通过本课题，我们发现SRM1649b的浓度及处理时间与细胞增殖呈现负相关；SRM1649b可抑制人成纤维及角质细胞细胞周期，使调控细胞周期的关键蛋白CyclinA和Skp2蛋白表达下调， 使细胞发生G1/S期及G2/M期阻滞；随着SRM1649b处理浓度的增加，细胞凋亡率明显的增加；SRM1649b可使胞浆内的芳香烃受体AhR显著转位至细胞核内。