原发性放射抗拒是喉鳞癌放疗失败的主要原因之一，其分子机制尚不清楚。本课题组在预实验中发现Spy1与喉鳞癌原发性放射抗拒相关，但机理不明。研究发现p19INK4d的磷酸化与DNA损伤修复密切相关，我们证实Spy1与p19INK4d相互作用并导致其磷酸化，因此推测Spy1通过介导p19INK4d的磷酸化参与喉鳞癌原发性放射抗拒。本课题拟应用1)免疫组化技术检测放射抗拒和敏感的人喉鳞癌标本中Spy1、p19INK4d的表达并分析其与放射抗拒的相关性；2）构建稳定过表达和干扰Spy1的喉鳞癌细胞株，检测改变Spy1的表达对p19INK4d的磷酸化状态的调控及DNA损伤修复的作用，并构建Spy1不同结构域的表达载体，明确Spy1与p19INK4d的作用位点；3）构建裸鼠成瘤动物模型，在体内进一步验证。本项目的成功研究将有助于丰富认识喉鳞癌放射抗拒的分子机制，并为临床探索喉鳞癌放射抗拒提供新的靶点。

Inherent radioresistance is a main cause of laryngeal carcinoma radiotherapy failure, but the molecular mechanism is not clear. In our previous study, we found the correlation between Spy1 and inherent radioresistance of laryngeal carcinoma. It has been reported that p19INK4d phosphorylation closely related with DNA damage repair. We verified Spy1 interacted with p19INK4d and caused p19INK4d phosphorylation, so we presumed that Spy1 could regulates the phosphorylation of p19INK4d involved in the inherent radioresistance of laryngeal carcinoma. In this study, we will use 1) the technology of immunohistochemistry detect the expression of Spy1 and p19INK4d in radioresistant and radiosensitive tissues of human laryngeal carcinoma, and analyze their correlation with radioresistance; 2) we will built stable cell line of Spy1 overexpression and interference, then detect the change of Spy1 expression impacts on the regulation of p19INK4d phosphorylation and DNA damage repair, in addition, we will built the expression vector of Spy1 different domain, verify the interaction site of Spy1 and p19INK4d ; 3) we further confirm that Spy1 regulates p19INK4d in the inherent radioresistance of human laryngeal carcinoma by xenografts in nude mice. The results of this study not only help us to enrich understanding of the molecular mechanisms of laryngeal carcinoma radioresistance, but also provide a new target to solve the radioresistance of laryngeal carcinoma for clinic.