基因修饰人脂肪干细胞复合静电纺丝分层组装支架材料构建仿生淋巴管复合体的研究

The ideal treatment for limb obstructive lymphedema is the transplantation of lymphatic vessels, but the source of the healthy lymphatic vessels is limited. The construction of biomimetic lymphatics is a hopeful alternative to solve this problem, and the structure of the artificial lymphatic tube is single and the biological function is poor at present. Thus building an good function bionic lymphatic vessel is an urgent problem to be solved. On the basis of the study on the differentiation conditions of lymphatic endothelial cells and the electrospun technology, The aim of this study is to obtain a large number of human adipose stem cells(ADSCs), which are induced to lymphatic endothelial cells stablely, by using gene interference technique, Combined with electrospinning and clicking chemistry technology, acquiring the layered scaffold that the inner layer was PLGA and collagen, and the outer layer was the PCL layer scaffold that modified TGF- beta 1.the scaffold co-culture with the genetically modified ADSCs to acquired the functional biomimetic lymphoid tissue complex that the inner layer is lymphatic endothelial cells and the outer layer is smooth muscle like cells. The implant will be graft in animals and investigated the treatment effect to the lymphedema. The purpose and significance of this study is to create a new way to promote the clinical application of artificial lymphatic vessels, and provide new ideas for the limited source of seed cells and the regeneration and repair of functional lymphatics.

肢体阻塞性淋巴水肿理想的治疗方法是淋巴管道的移植重建,但健康肢体淋巴管的来源十分有限，构建人工仿生淋巴管为解决这一难题带来希望。目前构建的人工淋巴管结构单一，生物学功能差，因此如何构建组织结构合理，生物学功能良好的仿生淋巴管是亟待解决的问题。在课题组前期研究淋巴管样内皮细胞分化条件及静电纺丝技术的基础上，本研究拟利用基因干扰技术获得大量稳定向淋巴管样内皮细胞分化的人脂肪干细胞，结合静电纺丝及点击化学技术，获得内层为PLGA以及胶原，外层为修饰TGF-β1的PCL支架材料，通过3D共培养获得内层为淋巴管样内皮细胞，外层为平滑肌样细胞的功能性仿生淋巴管组织复合体，并将仿生淋巴管植入淋巴水肿动物体内,研究其在阻塞性淋巴水肿治疗中的效果。本研究的目的与意义在于为促进人工淋巴管临床应用开辟新途径，为种子细胞来源受限、功能性淋巴管的再生与修复提供新思路。

淋巴管道的移植重建，恢复受损淋巴管道连续性是阻塞性淋巴水肿理想的治疗方法，目前构建的淋巴组织无法模拟淋巴管结构和功能，不能将生长因子向病变部位精准递送。课题组研究发现脂肪干细胞来源外泌体对淋巴内皮细胞存在调节作用并受转录因子Sox18的调控，而低氧状态能够增强外泌体的促淋巴管再生作用，为之后的淋巴管再生研究建立基础；通过膜融合技术，开发了一种可以高表达并递送的VEGFC工程化脂肪干细胞来源外泌体；通过近红外淋巴成像技术分析传统后肢淋巴水肿模型过快恢复的原因，以此为基础建立了改进的肢体淋巴水肿动物模型，能够更好地模拟人类淋巴水肿；以丝素（SF）和酪胺改性的明胶（GT）作为原材料，通过辣根过氧化物酶催化交联的方式结合，制备出兼具两者优点的SF/GT 复合水凝胶，并利用明胶水凝胶的温敏性和双氧水浸泡引发酶交联的特殊性，制备一种间隙可调的网状 SF/GT 水凝胶作为支架；覆盖工程化外泌体的水凝胶支架具有良好的生物相容性，能够在体外促进淋巴管再生，将其植入水肿部位能够实现病变部位精准递送，研究结果表明该材料能够在体内促进淋巴管再生，改善肢体淋巴水肿，为促进人工淋巴管临床应用开辟新途径。