人源性细胞外基质复合聚己内酯-明胶纳米纤维支架构建血液透析生物血管及其再生过程的研究

Hemodialysis artificial vascular grafts (AVG) are widely used. However, the elasticity or compliance of AVGs is quite different with natural artery. The AVGs are intolerant puncture and do not match with the anastomotic vessels which usually results in vascular stenosis and thrombosis. The main component of extracellular matrix in human being artery is collagen which exerts the tension and elasticity in the arterial wall. Xenogeneic extracellular matirx (ECM) exerts potential immunogenicity, but human being ECM is not readily available. So ideal dialysis AVG should be bionic human-being vascular structure and components. Hepatic B Virus (HBV) can activate the hepatic stellate cells (HSCs) to enhance the secretion of extracellular matrix (ECM) which plays a key role for the development of liver cirrhosis. So in this research, we will employ the ability of activated HSCs to prepare human-derived ECM biological vascular grafts. According to our previous study, the activated HSCs presented a stronger ability in secretion of collagen than fibroblasts, we will prepare polycaprolactone (PCL)-gelatin nanofibers vascular scaffold with electrospinning. We will seed HBV activated HSCs on the PCL-gelatin scaffold. The activated HSCs will secrete ECM such as collagen on the prepared vascular scaffold which will be cultured under self-made vascular bio-reactor. The ECM nanofibers remodels under the stress, and crosslinks with PCL nanofibers. At intervals, the ECM will be analyzed for an ideal production parameter. The HSCs-ECM-PCL vascular construction will be decellularized and lyophilized, and modified with heparin chitosan hydrogel for anticoagulant ability. The prepared biological vascular graft is sterilized with Co-60 radiation. The anticoagulant ECM-PCL vascular graft will be implanted for arteriovenous hemodialysis access in animal model. The analysis of the original ECM and PCL degradation, host immune and vascular cells infiltration, different layers of vascular wall formation or regeneration and host ECM regeneration will be carried out to confirm vascular mature duration and characters.

血液透析人工血管动静脉内瘘应用广泛，但其弹性和顺应性与人体动脉差异很大，不耐受反复穿刺，与吻合血管不匹配常并发狭窄血栓。动脉细胞外基质（ECM）中胶原蛋白是维持血管结构和力学性能的主要因素，理想的透析用人工血管应仿生动脉成分和结构。异种ECM丰富但有潜在抗原性，而临床上很难获得人ECM。乙肝患者因人肝星状细胞(HSCs)被病毒(HBV)持续激活而过量分泌胶原等ECM而发生肝硬化，本项目拟利用此特性制备具有人ECM成分的透析用生物血管，预实验证实HBV激活的HSCs分泌胶原的能力比成纤维细胞更强，我们将静电纺丝聚己内酯(PCL)-明胶纳米支架，再种植HBV激活的HSCs，动态培养下HSCs分泌胶原与PCL纳米纤维交联，检测ECM成分和性能以获得培养参数，脱细胞后用肝素-壳聚糖水凝胶修饰使血管有抗凝性，冻干及钴60辐射灭病毒。动物体内移植建立透析血管通路并检测成熟时间和性能，探索血管再生过程。

全球每年有近三百万肾衰竭患者做血液透析。血液透析人工血管动静脉内瘘应用广泛，但其弹性和顺应性与人体动脉差异很大，不耐受反复穿刺。同自体动静脉内瘘相比，人工血管与吻合血管不易匹配，常并发狭窄，易形成血栓，且感染率高。动脉的细胞外基质（ECM）中胶原蛋白是维持血管结构和力学性能的主要因素，理想的透析用人工血管应仿生动脉成分和结构。异种ECM虽然来源丰富，但有潜在抗原性，而临床上又很难获得人ECM。乙肝患者因人肝星状细胞（HSCs）被病毒（HBV）持续激活而过量分泌胶原等ECM而发生肝硬化，为本研究提供了思路。. 我们研发了一种人源性细胞外基质-聚己内酯-聚氨酯（ECM-PCL-PU）复合生物血管作为透析通路。通过静电纺丝获得具有抗凝作用的PU致密高分子纳米纤维层，在其表面进行PCL发泡获得多孔支架；再种植稳定转染HBX的肝星状细胞以分泌人源性细胞外基质；将血管置于反应器内，在应力下进行ECM塑形，再脱去细胞成分获得人纳米ECM-PCL-PU复合血管。移植该血管连接兔的颈动-静脉，检测血管、免疫细胞的迁移浸润，及血管细胞外基质成分在不同时相的动态变化，以探索该血管的再生重塑过程。. 我们制备的ECM-PCL-PU复合血管含有人细胞分泌的ECM，具有生物活体血管的天然结构和特性，有助于复合血管耐受透析时的反复穿刺；同时具有近似于天然血管的弹性和顺应性，弯曲成U型袢仍维持管状结构，不易打折。植入兔体内后，实验组（复合血管组）的血流通畅性、组织融合性、抗拉伸能力和细胞外基质再生情况等均优于对照组（单纯支架组）。. 我们研发的ECM-PCL-PU复合生物血管有望克服目前临床上使用的人工血管的缺点，为透析用血管的制备提供新的方法和思路，有着广泛的应用前景。