肛瘘的传统治疗方法创伤大、愈合时间长、肛门功能损伤发生率和复发率较高。利用同种异体脱细胞真皮基质（ADM）填塞治疗具有创伤小、愈合快、微创的优点，但成功率较低，感染被认为是失败的重要原因之一。为探索提高肛瘘治疗成功率的方法，本课题拟将纳米银整合到ADM上，在不改变ADM生物和物理性能的前提下，提高抗感染能力，探索纳米银的最佳抗菌浓度，以及纳米银-ADM的稳定性、细胞毒性、遗传学毒性和致癌性；建立不同浓度细菌污染的腹壁缺损模型，研究纳米银-ADM修补的可行性及对机体的影响；进一步建立肛瘘动物模型，采用组织细菌培养、免疫组化、western blot、实时荧光定量PCR和明胶酶谱法，期望研究纳米银-ADM填塞治疗肛瘘的可行性、对机体的影响，并初步阐明愈合机制。通过上述研究，期望为临床治疗肛瘘提供一种新的思路和研究方向。

The traditional treatment of anal fistula has lots of shortcomings, such as large trauma, long healing time, high risk of sphincter injury and high recurrence rate. Treatment with acellular dermal matrix (ADM) plug has the advantages of less trauma, faster healing, and minimally invasive, but clinical studies found that the anal fistula plug had relatively low success rate. Infection was considered to be one of the most important reasons for the failure. To further explore the method of anal fistula treatment, this project intends to firmly attach nano-silver to the ADM without changing the biological and physical properties of ADM, and aim to increase the resistance to infection, investigate the best anti-bacterial nano-silver concentration, as well as the stability, cytotoxicity, genetic toxicity and carcinogenicity of nano-silver-ADM composite material. To establish the contaminated abdominal wall defect animal model with different concentrations of bacterial, repair the abdominal wall defects with nano silver-ADM, and investigate the treatment efficacy, healing mechanism and effects on the body. To create the porcine model with fistula-in-ano and treate with nano-silver-ADM plug. By using bacterial cultures, Immunohistochemistry, western blot, real-time quantitative PCR and gelatin zymography, to evaluate the treatment efficacy, effects on the body, and the healing mechanism of nano-silver-ADM in the closure of anal fistula. Through the above study, expect to explore the feasibility of nano-silver-ADM plug treatment on anal fistula, and to clarify the healing mechanism, provide a new research thinking for the treatment of anal fistula.

本研究课题建立了纳米银-ADM复合材料，在不改变ADM生物和物理性能的前提下，提高抗感染能力，探索了纳米银的最佳抗菌浓度，以及纳米银-ADM的细胞毒性；建立不同浓度细菌污染的腹壁缺损模型，研究纳米银-ADM修补的可行性及对机体的影响；进一步利用纳米银-ADM填塞治疗肛瘘动物模型，采用组织细菌培养、免疫组化、western blot、实时荧光定量PCR等检测手段，研究了纳米银-ADM填塞治疗肛瘘的可行性、对机体的影响，并初步阐明了肛瘘的愈合机制。研究者通过本研究：1. 建立了纳米银-ADM复合材料，通过组织学、电镜和能谱检测证实未改变ADM的纤维结构，也未改变ADM的力学性能；纳米银-ADM材料对各种细菌具有一定抗菌性能；2. 通过与L-929细胞的共培养、MTT测定和扫描电镜检查发现，25ppm的纳米银-ADM的细胞毒性为1级，未影响细胞生长；3. 利用ADM和纳米银-ADM材料填塞治疗肛瘘，发现纳米银组在术后早期的肛瘘组织培养菌落数明显少于对照组；纳米银组新生血管生成时间较对照组更早，术后24h即出现；4. 通过免疫组化的检测发现，纳米银组的新生血管（VEGF）和MMP9高于对照组；而炎症因子（TNF-α）、抗炎因子（IκBα）和凋亡相关因子（HIF-1α）的表达则低于对照组。该结果在western blot 检测中得到进一步证实；5. 荧光定量PCR测定发现，纳米银组肛瘘标本中TGF-β1（促纤维化）、TGF-β3（抗纤维化）、Smad3（促纤维化）表达在术后1w-4w达到高峰，并高于对照组；两组的Smad7术后均逐步升高；通过上述研究，初步探索了纳米银-ADM 复合材料肛瘘栓治疗肛瘘过程中，通过抗菌、促进新生血管生成、促进组织纤维化、抑制炎症和凋亡等机制参与了肛瘘的愈合机制，为众多慢性感染性创面的治疗提供一种可供选择的方法。