PERK-eIF2α-ATF4信号通路介导的内质网应激在正畸牙周组织改建中的作用研究

During orthodontic treatment,how to control the orthodontic force applied onto the teeth,in order to ensure controllable and fast moving of them without threating their health? Orthodontic physicians have been plagued by this question for many years. What's the relationship between the loading modes and bone remodeling then? Only when we view this question through the biological and molecular response of human body to orthodontic forces,can we completely solve this problem...This study is based on the experimental research results gained by our group before, and we aim at examining the relationship between endoplasmic reticulum stress(ERS) response mediated by the PERK-eIF2α-ATF4 pathway and periodontal tissue remodeling during tooth movement in vivo and in vitro. The research will contribute to a better understanding of mechanism of bone remodeling during tooth movement, which may set the basis for clinical work. We intend to analysis the change of the expression of several related factors before and after force-loading from morphology, gene and protein level, in order to make certain the relationship between endoplasmic reticulum stress response mediated by the PERK-eIF2α-ATF4 pathway and periodontal tissue remodeling during tooth movement,and initially prove the mechanism of this signaling pathway to promote the action of osteogenesis, determine the critical point of the application of force, and set the basis for its clinical application.

在通过牙颌畸形的矫正实现颅面侧貌形态改善的过程中，如何控制正畸加力以实现牙齿健康可控而快速的移动，一直困扰着正畸医师。从机体对正畸力生物学反应的角度分析加力方式与牙周骨改建的关系，才能从根本上解决此问题。.本课题基于前期的研究，拟围绕机械力刺激引发内质网应激?激活PERK?eIF2α磷酸化?ATF4表达上调，最终促进PDLCs成骨分化，恢复内质网稳态展开，实验分体内、外两部分，通过构建大鼠和牙周膜细胞加力模型模拟临床正畸加力，验证内质网应激在正畸骨改建中的作用及机制；实验分别从形态学、基因及蛋白水平比较分析加力前、后成骨细胞标志基因、PERK-eIF2α-ATF4信号通路相关因子及内质网应激标志基因的表达变化，以分析内质网应激与正畸骨改建的联系；初步探明此条信号通路促进牙周组织改建的作用机理，确定其相应的加力临界点，为临床上实现牙齿快速移动奠定基础。

在前期实验中我们证实了转录激活因子4(ATF4)参与了正畸牙齿移动这一过程，在施加正畸力后ATF4的表达上调，进一步促进PDLSCs成骨分化。而蛋白激酶R样内质网激酶(PERK)在ATF4 的上游发挥作用。PERK是一种位于内质网膜上的I型跨膜蛋白，当细胞受到缺氧、钙离子平衡失调等刺激时，相应的信号通路将激活，引发内质网应激(ERS)。在ERS条件下，PERK被激活，导致真核细胞起始因子2α (Eukaryotic initiation factor 2α, eIF2α)磷酸化，产生一系列效应。关于内质网应激介导的PERK-eIF2α-ATF4信号通路在正畸牙齿移动中牙周膜干细胞成骨分化的作用还未见报道。.本研究通过人牙周膜干细胞的原代培养，构建牙周膜干细胞体外培养-力学刺激模型，并检测不同加力时间点后内质网应激相关因子和成骨相关基因的表达的水平变化，在此基础上，采用慢病毒质粒包装构建过表达、沉默表达PERK的稳定细胞系，对正常牙周膜干细胞和病毒转染的牙周膜干细胞实施应力刺激，研究ERS介导的PERK-eIF2α-ATF4信号通路在牵张力作用下牙周膜干细胞的成骨分化中过程中的作用。我们采用组织块法成功分离培养了hPDLSCs，hPDLSCs在加力后ERS相关因子(Bip, Xbp1, PERK, eIF2α, p-eIF2α)及成骨相关基因(ATF4,OCN, BSP)的表达基本上随加力时间延长而升高。利用慢病毒成功构建了过表达和沉默PERK的hPDLSCs模型，茜素红染色表明PERK过表达的hPDLSCs体外培养3周后可以观察到钙结节，而PERK干扰组几乎看不到钙结节。PCR和Western Blot结果显示，PERK过表达可以有效促进p-eIF2α, ATF4, OCN和BSP的表达，机械力刺激增加了这一效应，而PERK抑制则减少了这些因子的表达。.本课题证实了机械力刺激在一定程度上可以诱发ERS的发生，进一步激活PERK-eIF2α-ATF4信号通路。PERK过表达可以促进钙离子的沉积和基质矿化程度，促进牙周膜干细胞成骨分化；PERK呈现相反效应。ERS介导的PERK-eIF2α-ATF4信号通路参与了机械力作用下hPDLSCs成骨分化的过程，这为为进一步研究正畸牙槽骨改建的分子生物学机制提供科学依据，提高矫治效果提供新的思路和方向。