TSG-6对病理性瘢痕炎症反应的调控机制

Inflammation induced by wound healing can stimulate the migration of inflammatory cells, including mast cells, which promote the release of various vasoactive mediators and cytokines like TGFβ1, VEGF, which facilitate the proliferation of fibroblasts, the synthesis of type I and III collagen and neovascularization, leading to pathological scar formation. TSG-6 (tumor necrosis factor-stimulated gene-6), containing a highresolution Link module domain of hyaluronic acid (HA), has been shown to have anti-inflammatory activities. A significant reduction in TSG-6 levels has been noted within pathological scar compared with unscarred skin, but a significant increase compared with normal skin, as being consistent with the HA distribution, suggesting that TSG-6 might have the activities of anti-inflammation, anti-neovascularization and induction of apoptosis in pathological scar via inhibiting NF-κB/ IκB signaling pathway. Recombinant human TSG-6 is applied to the cultured fibroblasts isolated from the pathological scars and models of pathological scar in rabbit ears established, and then we will observe the vascular proliferation and histological changes, and detect the expression of inflammatory cytokines and NF-κB/IκB signaling pathway, microRNA regulation of fibroblasts and collagen synthesis, and apoptosis by TUNEL. This study is designed to provide new target, theoretical and experimental basis for the prevention and treatment of pathological scar. No relative studies have been noted.

创伤愈合时创面出现的炎症反应可促进肥大细胞等炎症细胞浸润,合成TGFβ1、VEGF等细胞因子,促进成纤维细胞增殖及I、III型胶原合成和血管新生,导致病理性瘢痕形成。已知TSG-6含有高度保守的HA结合区,具有抗炎作用，而病理性瘢痕组织中TSG-6表达较正常皮肤增高但较正常瘢痕组织显著降低,且与HA分布一致。由此推测TSG-6可抑制病理性瘢痕的炎症反应、抗血管新生及诱导细胞凋亡,其可能机制为阻抑了瘢痕NF-κB/IκB炎症信号通路。为此本研究采用TSG-6干预兔耳病理性瘢痕模型和体外成纤维细胞培养,观察微血管增殖抑制等组织学变化;高通量检测炎性因子表达及NF-κB/IκB信号通路的影响;成纤维细胞microRNA的调控及胶原合成;Tunel观察细胞凋亡。从不同角度观察其抑制炎症及成纤维细胞促凋亡的调控机制。本研究旨在为病理性瘢痕防治提供新的靶点、理论和实验依据。此类研究国内外未见报道。

病理性瘢痕是皮肤创伤后组织过度修复引起的以胶原等大量细胞外基质产生和沉积为特征的皮肤纤维化疾病。本病确切病因及发病机制仍未完全明确，已知与成纤维细胞过度增殖及胶原过度沉积有关。本项目探讨了TSG-6对病理性瘢痕的作用、对炎症反应及成纤维细胞增殖和凋亡的调控以及可能的分子机制和信号转导途径，以期为病理性瘢痕的治疗提供新的治疗靶点及理论和实验依据。经过四年的实验研究，本项目取得了以下重要进展：（1）成功体外分离培养病理性瘢痕成纤维细胞及正常皮肤成纤维细胞，体外与rhTSG-6蛋白共培养，通过MTT实验，流式细胞术，免疫组化法，Western blot实验，EMSA实验检测rhTSG-6处理后病理性瘢痕成纤维细胞的增殖凋亡情况以及rhTSG-6对NF-κB信号通路的影响；结果发现rhTSG-6处理组病理性瘢痕成纤维细胞增殖减少凋亡增加，病理性瘢痕成纤维细胞中异常激活的NF-κB信号通路受抑明显；（2）建立同源TSG-6稳定过表达及空白对照的病理性瘢痕成纤维细胞系,并在此基础上互为对照进行实验,以实时荧光定量 PCR(qPCR)和 Western blot 检测 p53、p21、cyclin D1、 CDK4、Fas、NF-κB、bcl-2、bax、caspase-3/8等基因表达改变,监测 TSG-6 基因在调节成纤维细胞增殖与凋亡过程中细胞内多条信号通路的改变。结果表明转染TSG-6表达载体的病理性瘢痕成纤维细胞凋亡率明显增高，qRT-PCR 结果表明TSG-6基因过表达，p21、bax、Fas、FasL、FADD、caspase3、caspase8和 p53基因及蛋白表达水平增加，而Cyclin D1 和 NF-κB表达减低，同时Fas/FasL信号通路上调激活；（3）构建兔耳增生性瘢痕模型，造模术后即日及术后5天，10天，15天注射TSG-6及等量 PBS 作为对照，于术后21，28，42天取材，以透射电镜观察及TUNEL法检测瘢痕组织成纤维细胞凋亡的变化,采用免疫组化法及实时定量聚合酶链反应(RT-qPCR)法检测炎症因子白细胞介素-1β(IL-1β),白细胞介素-6(IL-6)及肿瘤坏死因子-α(TNF-α)在各组中的表达,通过比较各组瘢痕指数(SEI)及采用免疫组化和Masson染色检测 I、III 型胶原表达的不同来评价瘢痕增生程度的差异，结果表明TSG-6