Exosome介导ALA光动力抑制皮肤鳞癌生长的分子机制研究

Squamous cell carcinoma (SCC) was treated by ALA-photodynamic therapy (ALA-PDT) and achieved satisfied outcomes, however, its mechanism remains unclear. According to our previous study,ALA-PDT enhances exosome secretion of SCC cell. Exosome secreted by ALA-PDT treated SCC cell suppressed proliferation of SCC cell in vitro and down-regulate the expression of ERK which is regulated by miR-124 .Exosome is thought to play important roles in intercellular communications. We hypothesize that exosome is very important for suppressing growth of SCC by ALA-PDT via mediating transfer of miR-124.In order to confirm the hypothesis, we have primary cultured mouse SCC cell in vitro. We will confirm that exosome secreted by ALA-PDT treated SCC cell can suppress growth of SCC. We will identify the microRNA in exosome, analyze the regulatory role and molecular mechanism of miR-124 in ALA-PDT suppressing growth of SCC by exosome .An insight view into the mechanism can throw lights on the new mechanism of ALA-PDT treatment of SCC, and provide new theoretical evidence on the clinical effect.

ALA-PDT能有效治疗皮肤鳞状细胞癌 (SCC)，但其机制仍不完全清楚。本课题组前期研究观察到ALA-PDT可以明显促进SCC分泌exosome，PDT后SCC细胞分泌的exosome可明显抑制SCC细胞体外增殖，下调细胞内ERK的表达。由于exosome是调节细胞间相互作用的重要媒介，鳞癌中ERK的表达受miR-124调控。由此推测，exosome可能通过转移miR-124，在ALA-PDT抑制皮肤肿瘤生长中发挥重要作用。为证实以上假设，本项目拟在已培养小鼠皮肤鳞癌细胞及前期研究基础上，进一步研究exosome在ALA-PDT抑制SCC生长中的作用及机制；在对exosome成分分析的基础上，重点研究miR-124的作用及分子机制。本项目的完成，有助于深入认识ALA光动力抑制皮肤鳞癌生长的机制，为提高ALA-PDT治疗SCC的临床疗效提供新的理论基础。

背景：ALA-PDT能够诱导抗肿瘤免疫，但对于ALA-PDT处理后鳞癌细胞分泌的exosome是否改变及其对肿瘤细胞和肿瘤微环境中其他细胞的作用尚未见报道。.目的：探究ALA-PDT对小鼠鳞癌细胞、成纤维细胞分泌的exosome的改变，并研究该exosome对鳞癌细胞和肿瘤微环境的作用。.方法：在已建立SKH-1无毛小鼠SCC (Squamous cell carcinoma)模型基础上，原代培养小鼠皮肤鳞癌细胞，提取及鉴定小鼠鳞癌细胞来源exosome，探究ALA-PDT对小鼠皮肤鳞癌细胞、成纤维细胞分泌exosome的影响。分别研究ALA-PDT处理的小鼠皮肤鳞癌细胞exosome对皮肤鳞癌细胞、成纤维细胞、树突状细胞的影响。.结果: 成功从小鼠皮肤鳞癌组织中获得皮肤鳞癌细胞，生长良好，连续传代。通过透射电镜，粒径分析、Western Blot检测exosome表面标志性蛋白鉴定，ExoQuick-TC试剂盒成功提取鳞癌细胞分泌exosome。ALA-PDT处理后的小鼠皮肤鳞癌细胞株、原代小鼠皮肤鳞癌细胞，小鼠成纤维细胞株来源的exosome浓度较未经ALA-PDT处理组exosome蛋白浓度升高。ALA-PDT处理鳞癌细胞来源的exosome对于鳞癌细胞无明显的增殖及杀伤作用。ALA-PDT处理皮肤鳞癌细胞来源的exosome无法促进或抑制成纤维细胞分泌IL-6、VEGF。但是可以促进成纤维细胞分泌TGF-β1。Exosome可以被小鼠骨髓来源未成熟树突状细胞所摄取，并可刺激其表型成熟及功能成熟。.结论：ALA-PDT可以促进鳞癌细胞及成纤维细胞分泌exosome。ALA-PDT后鳞癌细胞分泌的exosome可被鳞癌细胞和DC细胞摄取，对鳞癌细胞无直接杀伤作用，但可促进成纤维细胞分泌TGF-β1，同时可以促进DC细胞的表型和成熟。本研究通过不同角度提供了了ALA-PDT治疗皮肤鳞癌的免疫学新方向，为今后在ALA-PDT治疗鳞癌的免疫机制研究提供新思路。