周围神经损伤后再生速度很缓慢，一直是困扰临床的难题。FK506通过免疫抑制和神经营养促进周围神经再生的作用已被国内外多项研究证实。我们前期研究首次发现FK506还能通过诱导成纤维细胞凋亡来减少神经吻合口瘢痕形成而促进周围神经再生。但FK506诱导成纤维细胞凋亡的确切分子机制及其对瘢痕形成过程中细胞外基质的影响尚不十分清楚。本课题拟采用大鼠神经吻合口瘢痕模型，通过检测内质网应激关键分子CHOP、Caspase-12、JNK以及细胞外基质COL-1，3、MMPs、LH2b等表达的变化，观察内质网应激在FK506诱导神经吻合口瘢痕成纤维细胞凋亡和细胞外基质表达中的作用。并采用siRNA技术抑制CHOP和Caspase-12的表达，从正反两方面阐明FK506诱导成纤维细胞经内质网应激途径凋亡的确切信号通路，更深入揭示FK506减少神经吻合口瘢痕形成的机制，为促进周围神经再生提供新的方法和研究思路。

The neural regeneration and functional recovery after peripheral nerve injury are very slow, which has been a tough problem in clinical medicine. It has been demonstrated by many investigations that FK506 could promote nerve regeneration through its immune inhibition and neurotrophic activity. Our previous study primarily found that FK506 could promote peripheral nerve regeneration through inducing fibroblast apoptosis and reducing scar formation in nerve anastomosis. But the exact molecular mechanisms by which FK506 induces fibroblast apoptosis and the influence of FK506 on extracellular matrix in the process of scar formation remain unclear. Through establishing nerve anastomosis scar in rat model and detecting the expression levels of endoplasmic reticulum stress (ERS) related molecules (CHOP, Caspase-12 and JNK) and extracellular matrix (COL-1, COL-3, MMPs and LH2b et al), this study is to explore the effect of ERS on fibroblast apoptosis and extracellular matrix expression induced by FK506. Also, we will investigate the influence of CHOP siRNA and Caspase-12 siRNA on fibroblast apoptosis and extracellular matrix expression induced by FK506. Thus, through the research above-mentioned, we can elucidate the exact cell signaling of ERS by which FK506 induces fibroblast apoptosis and further reveal the mechanisms by which FK506 reduces scar formation in nerve anastomosis. Moreover, this study will provid new methods and ideas for promoting peripheral nerve regeneration.

研究背景：疤痕形成是由于成纤维细胞凋亡减少、过度增殖导致的，因此，促进成纤维细胞的凋亡是减少疤痕组织形成的关键因素。大量研究已经证实FK506能够减少体内的疤痕形成，并且FK506能够减少内质网应激，然而，FK506对内质网应激介导的成纤维凋亡的作用机制一直尚未清楚。研究方法：本课题中，我们利用小鼠的皮肤成纤维细胞进行相关研究。实验中，利用细胞计数试剂盒Kit-8检测细胞的活力，膜联蛋白V /碘化丙锭双染检测细胞凋亡，用siRNA或者慢病毒转染技术进行基因敲除，凋亡相关蛋白表达水平通过Western blot检测，利用免疫功沉淀技术检测蛋白质间的相互作用。研究结果：FK506能够显著降低成纤维细胞活力并且诱导其凋亡。实验中我们发现FK506能够激活内质网应激反应（ER stress），进一步研究表明FK506诱导的凋亡是通过CHOP激活的内质网应激反应（ER stress）实现的，并且内质网应激反应（ER stress）抑制剂TUDCA和CHOP阻断相关实验能够减少凋亡的发生也证实了这一现象，更深层次的研究，免疫功沉淀结果表明FK506能诱导FKBP12.6从RyR2分离，并且从内质网膜转移到胞质中，最终促进内质网应激反应（ER stress）介导的细胞凋亡。结论：FK506能够通过CHOP信号通路激活内质网应激反应（ER stress），从而介导成纤维细胞凋亡。