Survivin 在瘢痕疙瘩中的作用及机制研究

Over proliferation and apoptosis obstruction is one of the important mechanisms of keloid formation. As keloid fibroblasts (KFB) cells have some characteristics of tumor cells, how to inhibit production and induce apoptosis is the focus of keloid research. In recent years, survivin gene is found to play an important role in tumor apoptosis, but its role in keloid is not clear. Our recent experiments showed that compared with normal skin, survivin expression in keloid is obviously high, expression suppression of survivin could inhibit cell growth and induce apoptosis. Based on this, the research take KFB cells as the research object, firstly detect expression of survivin in keloid; and use survivin over expression vectors or survivin-shRNA carriers to observe the effect of them on KFB cell biology behaviors and related signaling pathways; finally animal model is applied to observe the effect of survivin inhibitor in vivo and its synergies with corticosteroid. The project try to discuss the role of survivin in keloid from cells, molecular, animal models and drug synergies levels, which is help to provides a new approach to explore the pathogenesis and treatment options of keloid.

瘢痕疙瘩形成的重要机制之一即为成纤维细胞的过度增殖和凋亡受阻，瘢痕疙瘩成纤维细胞（KFB）具有肿瘤细胞的某些特征，因此抑制其增殖、诱导其凋亡一直是研究热点。近年来发现survivin基因在肿瘤细胞凋亡中发挥重要作用，但其在瘢痕疙瘩中的作用尚不清楚。申请者最近实验表明：与正常皮肤相比，survivin在瘢痕疙瘩组织中高表达，抑制其功能可抑制KFB的增殖，并促进其凋亡。 本课题以此为基础，首先检测survivin的特异性表达，并应用 survivin过表达载体和survivin-shRNA载体观察对KFB细胞生物学行为的影响及相关信号传导通路；最后应用裸鼠瘢痕疙瘩模型，观察survivin抑制剂的体内作用效果及其与皮质激素的协同作用。本项目从细胞、分子、动物模型和药物协同等多层面探讨survivin基因在瘢痕疙瘩中的作用，将有助于为探索瘢痕疙瘩的发病机制和治疗理念提供新思路。

瘢痕疙瘩成纤维细胞（keloid fibroblasts,KFB）的过度增殖及凋亡受阻促进了瘢痕疙瘩的形成和发展，Survivin是目前发现较强的凋亡抑制因子，同时具有调控细胞周期和细胞凋亡的功能，并且促进细胞的异常增殖。本项目首先证实Survivin在瘢痕疙瘩组织呈阳性表达；其次构建survivin-shRNA、survivin（+）过表达慢病毒载体，分别转染KFB细胞，成功建立稳转survivin-shRNA/survivin-OE的细胞模型。而后，通过流式细胞术、细胞划痕实验、transwell实验等，检测细胞凋亡率、细胞周期、细胞运动侵袭能力的差异。结果示：与对照组相比，survivin- shRNA组细胞凋亡率升高，S期细胞数目百分比减少，G1期增加；运动侵袭能力明显下降；SURVIVIN-OE组凋亡率降低，S期细胞数目百分比增加，G2期减少；运动侵袭能力明显升高。利用western blot发现survivin-RNAi组细胞collagen-Ⅰ、MMP-9、2和PCNA的表达均明显降低，，Survivin-OE组上述蛋白表达增高，证实Survivin的高侵袭力和抗凋亡性与MMP-9、2和PCNA密切相关。最后，建立裸鼠瘢痕疙瘩模型，局部注射survivin-RNAi质粒，发现移植物体积缩小，实时定量PCR和western blot结果显示Survivin及Collagen-Ⅰ的表达明显降低。因此Survivin是促进瘢痕疙瘩KFB凋亡抵抗、细胞侵袭增高的重要因子之一。 本项目初步阐明了survivin基因在瘢痕疙瘩中的作用，对于丰富瘢痕疙瘩的基础理论和指导临床靶向治疗的研究具有现实和理论意义。