Connexin26对辐射诱导的皮肤损伤相关趋化因子CCL27分泌和转导的影响及机理研究

The radiation-induced skin damage is the most common complication in the patients with cancer radiotherapy. Severe radiation-induced skin damage may attenuate the efficiency of radiotherapy because of limited radiation dose. CC chemokine ligand 27 (CCL27) mediated skin immuno-inflammatory response plays important role in regulation radiation induced skin damage. The keratinocytes of epiderm, from which CCL27 is secreted, could specially express connexin26 (Cx26) that can form gap junction which play important role in mediating radiation damage effects. Previous studies find that Cx26 participate the production and transmission of radiation induced damage signals. And Cx26 also interact with MAPK pathways and NF-κB/COX-2 pathways, which take part in regulating the production and secretion of CCL27. These results indicate that Cx26 might regulate the radiation induced secretion of CCL27. In this study, we will investigate the effects of expression level of Cx26 on secretion of CCL27 after radiation in vitro. Meanwhile, we will try to find the interaction of Cx26 and key proteins that participate in the expression of CCL27, and undercover the mechanisms of the role of Cx26 in regulating CCL27. We will also study the relationship of Cx26 and radiation induced skin damage in mouse models to confirm the findings from in vitro studies. We hope that our results will help to understand the molecular mechanisms of radiation induced skin damage, and provide theory evidences for protection and therapy of radiation induced skin damage during radiotherapy.

放射性皮肤损伤是肿瘤放疗中最常见的并发症，严重情况下导致治疗中断，造成肿瘤控制率和治愈率降低。趋化因子CCL27介导的皮肤免疫炎症反应对调节放射性皮肤损伤具有重要作用。分泌CCL27的皮肤角质细胞能够特异性表达连接蛋白Connexin26（Cx26），而研究发现Cx26参与了辐射诱导损伤信号的产生和传导，并且Cx26能与调节CCL27分泌的MAPK和NF-κB/COX-2信号通路相互作用，提示Cx26可能调节辐射后CCL27的分泌。申请人将在体外细胞实验中，研究Cx26表达水平对辐照后CCL27分泌的影响；同时研究Cx26与CCL27表达通路蛋白相互作用，探索Cx26调控CCL27的机理。在动物模型中开展研究，建立Cx26表达与放射性皮肤损伤的关系。研究结果将有利于揭示放射性皮肤损伤的分子机理，为放射性皮肤损伤的防护和治疗提供理论依据。

辐射引起的皮肤损伤是癌症放射治疗患者最常见的并发症。趋化因子CCL27介导皮肤免疫炎性反应在调节辐射诱发皮肤损伤中起着重要作用。分泌CCL27的皮肤角质细胞（HaCaT）特异性表达可形成间隙连接的Connexin26(Cx26)，这是传输辐射损伤信号的重要方式之一。Cx26还与MAPK通路和NF-κB/COX-2通路相互作用，而后者参与调节 CCL27的产生和分泌。在本研究中，用不同剂量X射线进行照射HaCaT细胞，检测与Cx26相关的MAPK信号通路蛋白及分泌CCL27相关蛋白的表达情况，并通过克隆形成实验、微核实验和ELISA检测细胞辐射敏感性。通过小鼠模型实验，在动物个体水平上验证辐射引起的皮肤损伤中CCL27的分泌变化及对皮肤放射性损伤的影响。利用CRISPR/Cas9质粒构建敲除Cx26表达的HaCaTCx26-/-细胞，以检测Cx26表达水平不同对CCL27分泌及辐射敏感性的影响。结果发现，HaCaT细胞辐照后，MAPK和 NF-κB信号通路被激活，CCL27分泌增加。小鼠经过急性和累积照射后出现不同程度皮肤损伤和炎症，其损伤程度与CCL27表达呈增相关趋势。Cx26表达敲除的细胞HaCaTCx26-/-辐照后CCL27分泌增加，细胞存活率上升，微核形成率下降。检测发现MAPK和 NF-κB信号通路的激活水平明显低于HaCaT细胞。这些结果表明，Cx26可能参与了辐照后HaCaT细胞中MAPK信号通路和 NF-κB/COX-2信号通路的激活，诱导CCL27的分泌，从而影响皮肤角质细胞辐射敏感性。本研究结果对于Cx26在临床上的潜在应用，降低辐射诱导的皮肤损伤，提供了实验依据和理论指导。