生物响应性水凝胶复合脐血干细胞治疗消化道瘘的研究

Non-surgical therapy to accelate fistula healing and avoid second-time operation is one of the key issues for abdominal surgery. It is demonstrated from our preliminary study that adjuvant use of autologous platelet-rich fibrin gel (PRFG) facilitated fistula occlusion and shortened hospital stays. Recent data show that umbilical cord blood (UCB)-derived hematopoietic stem cells (CD34+) can promote the vascularization and healing.We hypothesize that co-culturing CD34+ cells along with endothelial cells (ECs) carried by biologically responsive hydrogels such as PRFG, injectablechitosan-hyaluronic acidbased hydrogels (CS-HA) could take advantage of mutual benefits, enhancing the therapeutic effects. In the present study, we aims to investigate the effects of hydrogels on stem cells growth, adhesion, proliferation, and differentiation, to explicit the mutual influences between fistula tracts and scaffolds, and also the clinical efficacy and safety of this cell therapy. Mini-MACS immunomagnetic separation, fluorescence-activated cell sorting, laser scanning confocal microscope, and pathological analysis would be used in this study from molecular, cellular, histological and whole aspects. The scaffold used in this study could become an effective treatment to promote fistula closure and shorten the courses of disease.

非手术疗法促进消化道瘘自行愈合，免除病人再次手术是腹部外科的一项重要课题。前期研究表明自体纤维蛋白胶(PRFG)封堵消化道瘘可加速瘘的愈合、减少住院时间；脐血干细胞可促进血管新生、加速创面愈合。我们提出以下假说，在生物响应性水凝胶PRFG、壳聚糖-透明质酸复合胶(CS-HA)中种植脐血来源CD34+细胞及其分化的内皮细胞(EC)，发挥两者的协同作用促进瘘的愈合，进一步提高胶堵治愈率。本研究以水凝胶作为干细胞载体，建立脐血CD34+细胞和EC共培养体系，构建细胞-生物支架复合物封堵消化道瘘。拟采用磁珠分选、流式细胞分析、激光共聚焦、组织病理学等手段，从细胞、组织以及整体水平探讨生物支架对干细胞生长、黏附、增殖、分化的影响，明确复合物与机体的相互作用，以及临床应用的疗效与安全性。本研究采用的生物响应性水凝胶-干细胞复合物将可能成为提高消化道瘘自行愈合率、缩短自行愈合时间的一项有效措施。

非手术疗法促进消化道瘘自行愈合，免除病人再次手术是腹部外科的一项重要课题。生物水凝胶可促进组织修复，与机体相互作用；脐血干细胞可促进血管新生、加速创面愈合。本项目首先合成生物响应性水凝胶人源单供体富血小板纤维蛋白胶(PRFG)和壳聚糖-透明质酸(CS-HA)，分析表征其理化性质；构建干细胞-水凝胶复合物，细胞分布在呈纤维网状的凝胶中。发现以CS-HA为载体的CD34+及其分化的内皮细胞共培养的细胞活力显著高于单独培养的CD34+细胞组，CD31、VEGF、vWF等内皮细胞抗体表达更高，并通过NFκB/ERK通路调控炎症反应；动物实验探讨了生物响应性水凝胶对创面愈合的影响，表现为水凝胶组细胞聚集和基质沉积更多，胶原纤维的数量和密度更高，微血管更多等；最后通过前瞻性临床试验评价了生物响应性水凝胶PRFG封堵消化道瘘的疗效与安全性，与对照组相比，凝胶封堵瘘可安全、有效地治疗低流量管状瘘。回顾治疗预后相关的危险因素，发现肺炎克雷伯杆菌是PRFG治疗失败的显著相关因素。本研究的成果应用于临床，提高了消化道瘘自行愈合率、缩短了自行愈合的时间。