细胞质膜钙泵(PMCA1)与钙网蛋白(CRT)正反馈调控介导持续功能矫形力诱发成肌细胞凋亡的机制研究

We have already proved that orthopedic mechanical force can induce myoblast apoptosis of maxillofacial skeletal muscle, which is orchestrated by CRT-mediated endoplasmic reticulum stress. But how could persistent stretch stimulation promote the overexpression of CRT and ER stress? Scholars agree that the level of CRT is closely related with calcium homeostasis. Plasma membrane calcium pump 1 (PMCA1) is one of the major calcium pump that could pump the calcium out of the cells. Our previous experiments have found that the intracellular calcium concentration is positively correlated with the duration of stretch; Besides, the level of PMCA1 was decreased in myoblasts under stretch stimulation. Based on some conclusions that PMCA1 and CRT could modulate the expression of each other, we speculated that the unregulated CRT level and downregulated PMCA1 level in stretched myoblasts could be promoted by the positive feedback mechanism between the two factors. The imbalance between the two calcium related protein lead to breaking down of calcium homeostasis. This finally result in the CRT-mediated endoplasmic reticulum stress and apoptosis of myoblasts. The present program intends to use some in vitro and in vivo techniques such as overexpressing/knocking down target genes and target genes knocking-out mice, in order to prove that the positive feedback mechanism between PMCA1 and CRT is critical in persistent stretch-induced apoptosis of myoblasts, and to explore the possible molecular mechanism. The current study could provide theoretical evidences and new ideas on clinical orthopedic treatment.

我们已经证明：功能矫形力作用下颌面部骨骼肌的成肌细胞凋亡受到CRT介导内质网应激的调控。但持续应力刺激如何导致CRT过表达和内质网应激？学者认为CRT的表达水平与胞内钙离子稳态密切相关。细胞质膜钙泵(PMCA1)是将胞内钙离子排出细胞的主要离子泵之一。预实验发现：成肌细胞中钙离子浓度与应力刺激作用的时间正相关；应力刺激导致成肌细胞中PMCA1表达水平下降。鉴于PMCA1与CRT可以通过不同的信号通路调控彼此的表达，据此推测：持续应力刺激作用下，成肌细胞中PMCA1表达的下降与CRT表达的升高通过正反馈调控而相互促进，造成二者平衡的打破与钙离子稳态的失衡，最终引起CRT介导下内质网应激和细胞凋亡。本项目拟采用细胞基因转染、基因敲除小鼠等体内外实验方法，旨在证明PMCA1与CRT的正反馈调控在持续应力刺激诱导成肌细胞凋亡中的重要作用，并探索其分子机制，为临床功能矫形治疗提供理论依据和矫治思路。

张应力刺激诱导成肌细胞凋亡机制的探索对于阐述面颌部骨骼肌在临床功能矫形治疗中的改建机理意义重大。本课题深入探索了成肌细胞在张应力刺激下的钙离子稳态改变对于细胞凋亡诱导的决定性作用，并发现钙离子结合伴侣CRT和细胞质膜钙离子泵PMCA1存在交互对话，张应力刺激通过激活CRT-PMCA1反馈调控而导致CRT表达升高，PMCA1活性降低，从而引起胞内钙离子过载。本项目详细开展了以下研究：细胞实验中，对体外培养的成肌细胞实施不同参数的张应力刺激，利用荧光定量PCR，Western Blot，免疫荧光染色，细胞流式等方法探索成肌细胞凋亡相关通路；动物实验中，构建动物下颌前伸面颌部肌肉牵张模型，观察骨骼肌在牵张后的组织变化与成肌细胞凋亡情况。研究结果发现：（1）体外培养的成肌细胞在张应力刺激下的凋亡与应力刺激的时间、力值成正相关；（2）胞内钙离子过载参与了张应力刺激诱导下的成肌细胞凋亡；（3）成肌细胞在张应力刺激下激活CRT-PMCA1反馈调控机制；（4）张应力刺激通过CRT-CaM途径抑制PMCA1活性，并通过PMCA1-p38MAPK途径促进CRT表达；（5）张应力刺激导致成肌细胞的钙离子稳态的变化与CRT-PMCA1反馈调控密切相关，并且基因敲除CRT或过表达PMCA1可以部分减弱应力刺激对钙离子稳态的影响，以及减少成肌细胞在应力刺激下的凋亡；（6）大鼠颌面部骨骼肌牵张模型中，骨骼肌纤维发生不同程度的改建，成肌细胞未发生明显凋亡。以上研究表明：体外培养成肌细胞在张应力刺激下发生凋亡与CRT-PMCA1反馈调控激活引起胞内钙离子过载有关；而在体内张应力环境中成肌细胞未发生明显凋亡的机制还有待进一步研究。本课题将拓展张应力刺激诱导成肌细胞凋亡的分子机制，并为临床功能矫形治疗的优化和改进提供实验证据。