Caspase蛋白酶在牵张应变诱导人牙周膜细胞凋亡中调控机制的研究

Excessive force loading to teeth by improper manufactured dentures or improper orthodontic treatment will result in periodontal tissue wound. There is cell death in the process of periodontal tissue wound and remodeling. Apoptosis is an important way of cell death. The response of periodontal ligament (PDL) cells to mechanical stimulation is important in periodontal tissue wound and remodeling. There are few reports about the relationship between force loading and apoptosis of PDL cells. Previously, we reported that cyclic stretching strain induced apoptosis in human PDL cells and caspases involved in the stretch-induced apoptosis. However, the mechanism involved in this process remains unclear. In the present research, we will probe into the mechanism of the caspases signaling involved in the stretch-induced apoptosis in PDL cells. The human PDL cells will be stretched by 20% strain for 6 and 24h. After being stretched, the expression of the activated caspase-3, -5, -7, -8 and -9 will be detected by Western blot. Human PDL cells with CASP3, 5, 7, 8 and 9 genes being silenced with respective shRNA will also be stretched and the apoptosis rate will be measured and compared with that of stretched cells without gene silencing. The interaction between these caspases will also be examined by stretching the gene silenced cells and detecting the activated caspases. The interaction between the caspase-5 and other apoptosis related signaling factors will be researched by real-time PCR array analysis and immunoprecipitation. The results of the present research may give theoretic supports to the oral physiological and oral pathological researches and give instructions to the proper force loading in the prosthodontic and orthodontic treatments and prevention of occlusal trauma and periodontitis.

义齿设计制作不当以及正畸矫正牙齿时施力不当，使牙齿受力过大，均可造成牙周损伤。在牙周损伤、改建中必然伴随相关细胞死亡，而细胞凋亡与细胞死亡密切相关。牙周膜细胞（PDLCs）在牙周损伤和牙周改建中具有关键作用，但力与PDLCs凋亡的关系研究甚少。申请者首次报道牵张应变能诱导人PDLCs凋亡，caspase蛋白酶参与了牵张诱导的细胞凋亡，但其具体调控机制尚不清楚。据此本课题拟运用免疫印迹技术、基因沉默技术等研究手段，检测caspase-3、-5、-7、-8、-9在牵张应变诱导HPDLCs凋亡过程中活性状态的变化，并测定相应基因沉默后牵张应变诱导HPDLCs凋亡率的变化，以及探索各蛋白酶之间的相互作用关系，探讨caspase蛋白酶在牵张应变诱导HPDLCs凋亡中的调控机制，以期为口腔生理学和病理学的基础研究提供相关理论支持，并为口腔修复及正畸临床对牙齿合理施力以及牙合创伤、牙周病的防治提供指导。

本项目采用自行研制开发的CSU-A型动态机械应变细胞加载装置对体外培养的人牙周膜细胞（human periodontal ligament fibroblast，HPDLCs）施加不同大小的动态牵张应变，并作用不同的时间段，运用免疫荧光技术、激光扫描共聚焦显微成像技术考察了动态牵张应变对HPDLCs的形态和排列以及细胞骨架的的影响，证明牵张应变可以使细胞形态、排列以及细胞骨架的形态、排列发生规律性的变化。在此发现基础上，采用基因芯片检测动态牵张应变加载前后细胞粘附及细胞骨架相关基因的差异表达，发现了多个力学响应基因，为后续研究提供了可能的介入点。运用免疫荧光技术、流式细胞术等研究手段，考察了动态牵张应变对HPDLCs凋亡的影响，证实牵张应变诱导牙周膜细胞发生凋亡，且具有时间和应变值依赖性。运用免疫印迹技术检测动态牵张应变作用下HPDLCs caspase-3、-5蛋白酶表达的变化，证明了caspase-5蛋白酶参与了HPDLCs力学信号转导。完成了CASP5 shRNA慢病毒载体的构建、筛选和包装，采用CASP5 shRNA慢病毒载体对HPDLCs进行感染，成功实现了对CASP5基因的沉默，且通过Western blot验证了对caspase-5蛋白表达的抑制效果，为后续实验提供了良好的基础。目前正在进行CASP5基因沉默后牵张应变诱导HPDLCs凋亡率以及caspase-3蛋白表达变化的研究。课题组已有1篇标注课题资助的学术论文被中文科技核心期刊录用，即将刊出，1篇论文已撰写完成并已投递SCI收录期刊，参加国际学术会议交流1次，负责人招收硕士研究生1人参与本课题研究攻读硕士学位。在本研究基础上，项目组获得了2013年上海市“浦江人才计划”项目资助课题“牵张诱导人牙周膜细胞凋亡中caspase蛋白酶调控机制的研究”（项目编号：2013D21，2013.9-2015.8，20万元），探索多个caspase蛋白酶在牵张诱导牙周膜细胞凋亡中的作用和调控机制，进一步揭示牵张诱导细胞凋亡的机制，以期阐明力对牙周组织的作用机理。