miR-206在腭帆张肌放射性损伤中的表达及机制研究

Secretory otitis media is a high incidence after radiotherapy of nasopharyngeal carcinoma (NPC ) complications . Prophase studies found tensor veli palatine (TVP ) the radiation damage to open led to the initiative in the eustachian tube dysfunction caused by secretory otitis media , main show is muscle damage source sex , but the mechanism is still not clear . As the skeletal muscle specific miRNAs are becoming a hot spot of research on muscle damage , including miR-206 and its regulation of target genes HDAC4 and SRSF9 role in skeletal muscle damage is particularly important . Thus , we hypothesized that in TVP radioactive damage , miR-206 through regulating SRSF9 , HDAC4 cause disorder of TVP repair and regeneration , causes the atrophy and fibrosis . Tree shrews applied to this project to establish animal model of secretory otitis media after radiotherapy and uses the tissue block repeatedly adherent cultivation method in TVP myoblast , by Lentivirus mediated transfection technique ,testing TVP after irradiation of SOD , MDA and miR-206 , HDAC4 ,SRSF9 expression level , and discusses the molecular mechanisms of TVP radioactive damage . For the development of precision TVP damage after radiotherapy treatment and improve the cure rate of secretory otitis media after radiotherapy is of great significance .

分泌性中耳炎是鼻咽癌放疗后发生率较高的并发症。前期研究发现腭帆张肌（TVP）的放疗损伤导致了咽鼓管的主动开放功能障碍引起分泌性中耳炎，主要表现为肌源性的损伤，但机制仍未明确。随着骨骼肌特异性miRNAs成为研究肌肉损伤的热点，其中miR-206及其调控的靶基因HDAC4及SRSF9在骨骼肌损伤修复中的作用尤为重要。由此，我们提出假设：在TVP放射性损伤中，miR-206通过调控SRSF9、HDAC4引起TVP的修复、再生障碍，导致其萎缩及纤维化。本项目拟应用树鼩建立放疗后分泌性中耳炎的动物模型并采用组织块反复贴壁法分离培养TVP成肌细胞，通过慢病毒介导的转染技术，检测照射后TVP中SOD、MDA以及miR-206、HDAC4、SRSF9的表达水平，深入探讨TVP放射性损伤的分子机制。对于发展精准的TVP放疗后损伤的治疗方法以及提高放疗后分泌性中耳炎的治愈率具有重要意义。

放疗后分泌性中耳炎是鼻咽癌患者放疗后常见的并发症，其发生机制可能是放疗射线损伤腭帆张肌，引起咽鼓管主动开放功能障碍，最终导致放疗后分泌性中耳炎的发生。miR-206是一种骨骼肌特异性miRNA，在骨骼肌机械和化学损伤中通过调控HDAC4参与修复再生过程，但是miRNA是否调控HDAC4作用于骨骼肌放射性损伤的修复再生过程尚不明确。本次课题利用树鼩构建放疗后分泌性中耳炎动物模型，观察腭帆张肌放射后病理改变及放射后miR-206、HDAC4表达情况；研究不同剂量放疗后，成肌细胞在不同时间点miR-206、HDAC4表达情况；通过引入外源性miRNA探讨miR-206对成肌细胞的作用。结果显示：（1）对树鼩腭帆张肌放射后6个月，树鼩可发生分泌性中耳炎；（2）放射后腭帆张肌发生氧化损伤，毛细血管密度降低等，逐渐出现纤维化；（3）放射后腭帆张肌miR-206表达下调，HDAC4表达升高，HDAC4蛋白表达增加；（4）对成肌细胞不同剂量放射，在同一时间点4Gy放射后miR-206下降最明显；同一剂量放射后2小时miR-206下降最明显。（5）在同一时间点8Gy放射后HDAC4上调最明显；同一剂量放射后48小时HDAC4上调最明显。通过本次研究，我们发现在腭帆张肌放射性损伤中，miR-206 通过调控SRSF9、HDAC4 引起TVP 的修复、再生障碍，导致其萎缩及纤维化。