Shikonin诱导成纤维细胞凋亡在增生性瘢痕修复中的机制研究

Scarring is a significant medical burden; financially to the health care system and physically and psychologically for patients. While research effort has been directed at the development of improved approaches to remediate scars, currently available therapies are not satisfactory due to their undesirable side-effects, complex delivery routes, requirements for long-term use and/or expense. Therefore, a novel scar remediation therapy that is simple to use and has minimal side-effects and cost is of great importance for both clinicians and patients.Shikonin, an active component extracted from Zi Cao has been demonstrated to induce apoptosis in cancer cell lines. This is of interest since apoptosis is known to be an essential component of scar tissue remodelling. This has led us to hypothesise that perhaps Shikonin may induce apoptosis in hypertrophic scar-derived human skin fibroblasts and may therefore hold potential for scar remediation. Our recent studies have revealed that Shikonin does indeed induces apoptosis in human scar-derived fibroblasts. The overall goal of this project is to verify Shikonin induces apoptosis in cells in scars using sophisticated 2-D and 3-D in vitro cell culture approaches, and a large animal in vivo hypertrophic scar model， as well as the underlying mechanisms invoked in the process of apoptosis using Western Blot, RT-PCR,signaling pathway inhibitors and RNA interference. Completion of these studies will generate the critical information necessary to developed into cost- and therapeutically-effective novel scar therapies.

瘢痕是烧伤及创伤后常见的并发症, 寻找新型疗法迫在眉睫。Shikonin，紫草的有效成分，被证实诱导肿瘤细胞凋亡, 但是否诱导瘢痕细胞凋亡仍未知。鉴于细胞凋亡在瘢痕组织重塑中的作用, 我们假设Shikonin诱导瘢痕成纤维细胞(HSF)凋亡而具修复增生性瘢痕的潜力。我们最近的研究发现Shikonin诱导HSF凋亡, 且对正常角质形成细胞没影响。根据以上发现, 我们将运用细胞共培养系, 人体皮肤瘢痕等效模型及猪增生性瘢痕模型继续验证Shikonin诱导HSF凋亡及在增生性瘢痕修复中的作用。此外，本课题将采用Western Blot，RT-PCR，信号通路阻断剂, RNA干扰等技术, 研究两条经典凋亡通路（TNF-R1/Fas，Bcl-2介导的凋亡通路）与Shikonin诱导HSF凋亡的关系。 阐明Shikonin诱导HSF凋亡的信号转导机制和作用靶点，对开发新的瘢痕药物具有重要意义。

瘢痕是烧伤及创伤后常见的并发症, 现行瘢痕疗法因其副作用大、给药途径复杂、使用周期长且费用昂贵等，而不尽人意。经济有效、简单易用、副作用小的瘢痕修复疗法，迫在眉睫。Shikonin是从紫草中提取的有效成分，被证实具有多种生物活性，例如抗肿瘤、抗菌等。大量研究显示Shikonin诱导肿瘤细胞凋亡。鉴于细胞凋亡在瘢痕组织重塑中的作用, 我们假设Shikonin诱导瘢痕成纤维细胞（HSF）凋亡而具修复增生性瘢痕的潜力。.我们构建了猪烧伤增生性瘢痕模型，测试Shikonin是否促进瘢痕修复。结果显示Shikonin治疗瘢痕八周后, 瘢痕变为扁平化, 收缩少, 粉红色, 质地明显优于对照组生理盐水。H/E染色显示增生性瘢痕的表皮角质层变薄，角质细胞数量减少，而真皮中成纤维细胞数量减少。免疫组化显示Shikonin明显抑制增生性瘢痕组织中P63、CK10、α-SMA、TGF-β1和 Collagen I蛋白的表达，从而促进瘢痕修复。明确Shikonin具备修复瘢痕的作用后，体外细胞培养被用来揭示Shikonin促进瘢痕修复的分子机制。体外原代皮肤细胞单培养研究发现Shikonin诱导HSF凋亡, 且不影响角质细胞（HSK）。Western Blot显示Shikonin诱导HSF的ERK1/2和p38α/β蛋白磷酸化增加；减弱Bcl-2蛋白的表达；激活caspase 3蛋白表达并伴随着pro-caspase 3蛋白的出现。qRT-PCR显示Shikonin抑制HSF的COL1A1, COL3A1 和αSMA基因的表达。体外原代皮肤细胞共培养研究发现Shikonin抑制HSF增殖而不影响HSK。Western Blot显示Shikonin 诱导ERK1/2和JNK1/2蛋白的磷酸化增强；诱导Bcl-2蛋白的表达减弱；caspase 3蛋白被激活伴随着pro-caspase 3蛋白的出现。qRT-PCR显示Shikonin抑制HSF的CASP3, BCL-2, COL1A1, BAX, CYCS, COL1A1, COL3A1和α-SMA基因的表达。Shikonin诱导成纤维细胞凋亡信号通路为Erk1/2&p38/ JNK1/2→Bcl-2→Bax→Cycs→Caspase-3→细胞凋亡。综上所述，Shikonin诱导HSF凋亡，减少胶原蛋白表达，因而具有修复瘢痕的潜力。

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