增生性瘢痕的自噬作用及p53对自噬调节机制的探讨

The cellular homeostasis between cell proliferation and apoptosis contributes to the pathogenesis of hypertrophic scar (HS) in wound healing. Autophagy, as a new type of cell apoptosis, plays an important role in pathogenesis of several diseases in mammals. Our previous study showed that the mRNA and protein expression levels of LC3 and beclin 1 in HS were significantly lower than that in normal skin, and confirmed that the autophagic capacity in HS was repressed. However, the effect of cellular autophagy on HS formation and the molecular mechanism of autophagy are poorly understood. Published reports and our study have shown that cellular autophagy could be regulated by p53, but its regulatory mechanism is unclear by now. Based on these, our purposes, in this study, are ① to analyze the mRNA and protein expression levels of some key autophagy related genes and p53 in HS tissues and tissue-derived cultured fibroblasts, ② to explore the regulatory mechanism of p53 in autophagic target genes, ③ and to validate the autophagic capacity and the regulatory mechanism of p53 in rabbit ear HS model. The study will be able to explain that the depressed autophagic capacity is one of key factors of HS formation, elucidate autophagic regulatory mechanism by p53 in HS, and provide the theoretical guidance for HS prevention, treatment and drug screening.

伤口愈合过程中细胞增殖与凋亡的失衡是增生性瘢痕形成的重要原因之一，而自噬作为一种新的细胞凋亡方式，在多种疾病的发生过程中具有重要作用。前期我们的研究表明增生性瘢痕组织中自噬基因LC3和Beclin1的mRNA及蛋白表达水平明显降低，证实了在增生性瘢痕组织中细胞的自噬功能受到显著抑制，而自噬在增生性瘢痕发生过程中所起的作用及其作用机制目前尚缺乏研究。文献及我们的研究表明p53对细胞自噬具有重要调节作用，但p53对自噬的调控机制尚未明了。基于此，本研究拟 ①分析增生性瘢痕组织及离体培养细胞中关键自噬基因、p53基因及蛋白的表达水平；②探讨p53对细胞自噬靶点基因的调节机制；③利用兔耳瘢痕模型验证瘢痕组织的自噬功能及p53对细胞自噬的调节作用。该研究将明确自噬功能减弱是增生性瘢痕形成的一个关键因素；初步阐明p53对瘢痕组织中细胞自噬的调控机制；为增生性瘢痕的预防、治疗及药物筛选提供理论指导。

增生性瘢痕（hypertrophic scar，HS）是一种严重的皮肤纤维化疾病，其特征是过度的细胞增殖以及胞外基质的过度沉积，成纤维细胞的非正常激活是增生性瘢痕产生的重要病理性原因。前期我们的研究证实增生性瘢痕成纤维细胞（HS fibroblasts，HSFs）自噬功能异常与HS的形成及纤维化密切相关。但是哪些分子，如何调节HSFs尚不明确。为此，我们研究野生型p53（wtp53）对如何通过自噬性HSFs细胞凋亡抵抗HS的形成及纤维化。Western blotting结果表明：wtp53过表达（Adp53）能够明显促进感染HSFs的自噬活性，抑制胶原蛋白Col 1，Col 3及α-SMA的表达。而LC3过表达（AdLC3）对Col 1，Col 3及α-SMA的表达均无影响，相反，沉默LC3（shLC3）能够明显抑制HSFs细胞中Col 1及Col 3的表达，而不影响α-SMA的表达。Adp53调控的自噬表明依赖于Bcl-2和Bcl-xL分子, 而调控的自噬AdLC3仅仅依赖于分子Bcl-xL。流式细胞结果显示沉默Bcl-2（sibcl-2）及Bcl-xL（sibcl-xL）能够显著增加HSFs细胞的凋亡。因此，HSFs修饰的自噬及自噬性凋亡经由Bcl-xL来抑制胞外基质的的沉积。重要的是：注射重组腺病毒Adp53, AdLC3, shLC3及干扰RNA的sibcl-2，sibcl-xL均都能够改善兔耳瘢痕模型的外观、胶原排列的结构，减轻瘢痕的形成。这些结果进一步证实修饰HSFs细胞自噬及自噬性HSFs细胞凋亡经由分子Bcl-xL。可见，Bcl-xL可以作为HS治疗的潜在分子靶点，成为新的治疗策略。