调节TRADD信号通路对成纤维细胞转分化的影响及对食管术后狭窄的防治研究

In the process of wound healing, fibroblasts transformed into myofibroblasts and hypertrophic scar fibroblasts. The hyperplasia and persistence of myofibroblasts and hypertrophic scar fibroblasts can create scar tissue hyperplasia and contracture. Control fibroblasts differentiation, Inhibit the proliferation of myofibroblasts and hypertrophic scar fibroblasts is the key to reducing scar formation. Anastomotic stenosis and ESD postoperative stricture are common complication after operation of esophagus, and affect the quality of life seriously. There is no good method of prevention and treatment. Our study shows that TRADD gene lentiviral transfection can selectively inhibit hypertrophic scar fibroblast proliferation, promote apoptosis，inhibit esophageal tissue hyperplasia. We speculate that TRADD gene was transferred into normal fibroblast cells could inhibit the fibroblasts excessive transforming into myofibroblasts and hypertrophic scar fibroblasts.TRADD gene was transferred into the fibroblasts in phases of wound healing can promote myofibroblasts or fibroblasts apoptosis, inhibit scar, prevent and treat stricture after esophageal operation. We will use the TGF- β 1 to stimulate fibroblasts, and observe the effect of TRADD gene transfection on the cell phenotype transformation, proliferation. We use the chemical and immune coupling method to coat TRADD gene in esophageal stent and use stent as the vector of TRADD gene to study the effect of TRADD gene intervention on the prevention and treatment of postoperative esophagus stenosis and their mechanism.

在创面修复过程中，成纤维细胞转化成肌成纤维细胞和瘢痕成纤维细胞，其过度增生可造瘢痕和挛缩。控制成纤维细胞转分化，抑制肌成纤维细胞和瘢痕成纤维细胞增殖是减少瘢痕形成的关键。吻合口和ESD术后狭窄是食管术后常见并发症，严重影响生活质量，目前无有效预防治疗措施。我们的研究显示TRADD基因慢病毒转染能选择性抑制增生性瘢痕成纤维细胞增殖，促进凋亡，抑制组织增生。我们推测将TRADD基因转入正常成纤维细胞能抑制其向肌成纤维细胞或瘢痕成纤维细胞过度转化，将TRADD基因转入修复阶段的成纤维细胞可促进肌成纤维细胞或瘢痕成纤维细胞凋亡，抑制瘢痕的发展，可预防和治疗食管术后瘢痕狭窄。我们将用TGF-β1刺激成纤维细胞，观察TRADD基因慢病毒转染对其细胞表型转化、增殖等的影响。用化学及免疫偶联法将TRADD基因涂层食管支架，以支架为载体对模型进行TRADD基因干预，观察其对食管术后狭窄的预防治疗作用及其机制

在创面修复过程中成纤维细胞转化肌成纤维细胞和瘢痕成纤维细胞，其过度增生可造瘢痕和挛缩。控制成纤维细胞转分化，抑制肌成纤维细胞和瘢痕成纤维细胞增殖是减少瘢痕形成的关键。前期研究显示 TRADD 基因慢病毒转染能选择性抑制增生性瘢痕成纤维细胞增殖，促进凋亡抑制组织增生。我们推测将 TRADD 基因转入修复阶段的成纤维细胞可促进肌成纤维细胞或瘢痕成纤维细胞凋亡，抑制瘢痕的发展，预防和治疗食管术后狭窄。研究内容（1）TGF-β1 刺激L929细胞，检测L929细胞转分化情况；（2）构建具有生物活性的TRADD基因慢病毒，将 TRADD 基因转入TGF-β1 刺激的L929细胞，观察 TRADD 基因慢病毒转染对其细胞表型转化、增殖等影响；并在不同时间点进行TRADD基因的转染，明确最佳转染时间点；（3）建立ESD术后狭窄犬模型；以食管支架作为载体携带 TRADD基因慢病毒至ESD术后段，观察其预防狭窄效果；（4）探讨生物蛋白胶-激素缓释系统对食管狭窄的预防效果。重要结果（1）TGF-β1成功诱导L929细胞转分化，同时促进细胞增殖和胶原的分泌；（2）TRADD基因慢病毒转染后，细胞能稳定地高表达TRADD基因；过表达TRADD基因可抑制TGF-β1刺激的L929细胞增殖、胶原的分泌，促进细胞的凋亡；在48h进行转染，其抑制作用最为明显；（3）共完成犬食管ESD术46例，存活于术后6周共33例；以食管支架携带TRADD基因至犬食管ESD手术段，从临床干预效果看，狭窄率、HE染色体现的纤维化程度、各相关蛋白表达量差异不明显；（4）ESD术后局部喷涂生物蛋白胶和曲安奈德，对术后食管狭窄有一定的预防作用，且术后手术部分并无明显组织增生现象。术后创面喷洒生物蛋白胶/生物蛋白胶+激素，或术后2周安置TRADD基因涂层支架对临床内镜下预防和治疗食管术后狭窄有重要指导作用。