内质网应激在半骺板阻滞术调节骺板软骨内生骨化中的作用及机制研究

Hemiepiphysiodesis, which provides gradual deformity correction, is widely accepted for the management of limb angular deformity in skeletally immature patients with various implant technique. We have found it was hemiepiphysiodesis- induced asymmetric compressive load that acted on the epiphyseal plate and regulated the rate of endochondral ossification. After load removal, catch-up growth was occurred in the eiphyseal plate. Previous studies showed that endoplasmic reticulum stress had a potentially pivotal role to play in the regulation of endochondral ossification. We also have found that the loading and subsequent release induced the change of chondral apoptosis index, as well as the expression of CHOP and P65. We speculate that endoplasmic reticulum stress and NF-κB singnal pathway was activated by compressive load in the process of hemiepiphysiodesis. We plan to observe and measure the epiphyseal matrix synthesis, the rate of growth by endochondral ossification, the composite of resting zone, proliferative zone and hypertrophic zone, the maxtrix-related and endoplasmic reticulum stress-related protein expression, in a miniature pig hemiepiphysiodeseal model. We plan to find the relationship between the change of outcome and compressive load intensity; explore the mechanisms of compressive load- induced retardation of endochondral ossification and catch- up growth after load removal; and test the role of ERs and NF-κB singnal pathway in the process endochondral ossification by inhibited them. The study will comprehensively demonstrate the mechanisms of angular deformity correction and deformity recurrence with hemiepiphysiodesis , providie novel targets for the therapy of angular deformity correction.

半骺板阻滞技术是矫正儿童肢体成角畸形的首选。我们前期的工作发现该技术通过不对称的压应力作用于骺板，调节骺板软骨内生骨化的速度,应力释放后出现“追赶生长”的现象；应力加载和释放的过程伴随骺板软骨细胞的凋亡指数、CHOP和P65等蛋白表达的改变。我们推测，压力调节骺板软骨内生骨化与内质网应激和NF-κB 信号通路有关。本课题拟沿用已成功制备的小型猪半骺板阻滞动物模型，使用免疫组化染色、Real time PCR,、Western blot技术，观察不同时间点和部位骺板内生骨化的速度，静息层、增殖层、肥大层形态构成，基质及ERs相关蛋白、mRNA的表达的变化情况，研究其与应力变化的关系，探讨压应力阻滞骺板内生骨化和应力解除后“追赶生长”的机制；通过阻断ERs、NF-κB通路观察其在应力介导的软骨内生骨化中的作用。本课题对进一步阐明半骺板阻滞术、术后畸形复发的机制，为矫正畸形提供新思路有重要意义。

内质网应激（endoplasmic reticulum stress, ERs）在骺板软骨细胞增殖、分化，调节内生骨化的过程中起了重要作用。我们前期的试验也已证实在半骺板阻滞过程中，内质网应激相关蛋白表达明显升高。本课题属于前期课题的延续，主要研究的目的为两个：1）探讨阻滞过程中、阻滞解除后，不同的时间点、不同的应力作用部位内生骨化速度、凋亡指数、ERs相关蛋白表达、“追赶生长”现象与时间，应力强度变化的对应关系。2）探讨ERs及相关通路在半骺板阻滞介导的不对称应力调节骺板内生骨化过程中的作用。本研究证实了压应力强度、骺板厚度、基质相关的蛋白表达、内生骨化速度、“追赶生长”现象之间存在相关性。半骺板阻滞过程中，骺板厚度的逐渐变化与不对称应力分布有关，通过免疫组化染色及免疫印迹揭示此种凋亡由内质网应激所介导。而当阻滞解除以后，凋亡细胞，内质网应激和自噬相关标记的下调，而此时IκB/NF-κB通路被激活。我们证实了机械应力降低了骺板的生长率。内质网应激介导的凋亡和自噬和此过程相关，IκB/NF-κB通路的激活和应力解除后的复发有关，而使用4-PBA干预半骺板阻滞术引起的内质网应激可以降低增殖层骺板细胞的凋亡。