人脂肪干细胞来源细胞外囊泡促进组织工程再生膀胱血管化的研究

Deficiency in angiogenesis is the key factor that hinders the clinical therapeutic effects of tissue-engineered bladder augmentation. There are still drawbacks for stem cell therapy to promote tissue-engineered regenerated bladder angiogenesis. The applicator’s previous research results have proved preliminarily that human adipose stem cells (ASCs)-derived extracellular vesicles (EVs) are able to promote regenerated bladder angiogenesis. This plan will fabricate and optimize human ASCs-EVs-encapsulated tri-layer hydrogel composite scaffold to repair bladder defects in a rat model of tissue-engineered bladder augmentation. This plan aims to evaluate the feasibility of human ASCs-EVs in promoting angiogenesis to support morphological restoration and functional reconstruction of regenerated bladder. In particular, the crucial role and detailed regulatory mechanisms of microRNA-126 from human ASCs-EVs activating SDF-1α/CXCR4 pathway in vascular endothelium during angiogenesis will be explored and testified. This plan fabricates hydrogel composite scaffold creatively to solve the difficulties in applying hydrogel to bladder repair. Then, it combines human ASCs-EVs with the tri-layer scaffold innovatively, which inspires a brand-new therapeutic approach to overcome the bottleneck of bladder augmentation. Meanwhile, this plan discusses the detailed mechanisms of how human ASCs-EVs promote regenerated bladder angiogenesis, and finally lays the theoretical and technological foundation for complete morphological restoration and functional reconstruction of tissue-engineered regenerated bladder.

血管化不足是制约组织工程膀胱扩大术临床疗效的关键因素。干细胞疗法促进组织工程再生膀胱血管化仍存在缺陷。申请人前期研究结果已初步证实人脂肪干细胞（ASCs）来源细胞外囊泡（EVs）能够促进再生膀胱血管化。本项目将制备并优化包裹人ASCs-EVs的三层水凝胶复合支架，用于修补大鼠组织工程膀胱扩大术模型中的膀胱缺损，评价人ASCs-EVs通过促进血管化从而支持再生膀胱形态恢复和功能重建的可行性，着重探索并验证人ASCs-EVs中microRNA-126激活血管内皮SDF-1α/CXCR4信号通路在其中发挥的关键作用以及具体调控机制。本项目创造性地制备了水凝胶复合支架，解决了水凝胶用于膀胱修复的难点，进而创新性地结合人ASCs-EVs，为突破膀胱扩大术瓶颈确立了新型治疗手段，同时探讨了人ASCs-EVs促进再生膀胱血管化的具体机制，为最终组织工程再生膀胱完全形态恢复和功能重建奠定了理论和技术基础。

血管化不足是制约组织工程膀胱扩大术临床疗效的关键因素。干细胞疗法促进组织工程再生膀胱血管化仍存在缺陷。申请人提取并鉴定人脂肪干细胞（ASCs）来源细胞外囊泡（EVs），证实人ASCs-EVs能够显著促进血管化，其有效作用浓度为5×10^8/mL。申请人创造性地制备了膀胱脱细胞基质（BAMG）-氧化海藻酸（ADA）/明胶（Gel）-丝素蛋白（SF）水凝胶复合支架，结合水凝胶疏松多孔的优势，通过BAMG构建阻隔尿液的疏水层，利用SF增强整体力学强度，进而创新性地包裹人ASCs-EVs，而不影响整体弹性模量和最大拉伸强度。该复合支架通过局部释放人ASCs-EVs，显著促进再生膀胱血管化，再生血管密度和平均血管直径均接近正常水平。除了促进血管化之外，人ASCs-EVs有效缓解尿路上皮增生，促进神经再生，但神经再生密度仍然低于正常水平。人ASCs-EVs也被证实促进再生膀胱恢复正常膀胱轮廓外形，促进水凝胶降解，以及降低再生膀胱组织炎中性粒细胞和巨噬细胞浸润。经过该策略修复的再生膀胱能够有效扩大膀胱容量，并保持顺应性等尿流动力学参数与正常膀胱类似。包裹在该复合支架上的人ASCs-EVs激活膀胱CXCR4/SDF-1α信号通路，促进下游ERK1/2蛋白磷酸化，以及增加血管内皮生长因子（VEGF）分泌。人ASCs-EVs的促进血管化显著优于其来源的干细胞，并被证实与CXCR4/SDF-1α信号通路及其下游ERK1/2蛋白磷酸化密切相关。人ASCs-EVs被人脐静脉内皮细胞（HUVEC）摄取具有时间依赖性。在共培养条件下，相比于干细胞，富集提纯后的人ASCs-EVs更能有效地被HUVEC摄取，从而发挥优于干细胞的促进血管化能力。人ASCs-EVs被进一步证实通过传递微小RNA（miR）-126，抑制RGS16调节蛋白，激活HUVEC中CXCR4/SDF-1α信号通路，促进下游ERK1/2蛋白磷酸化从而增加VEGF分泌，最终促进血管化。本项目在既往干细胞研究基础上，首次将干细胞来源EVs应用于膀胱再生领域；并通过构建三层复合支架，解决了水凝胶应用于膀胱全层修复的难点；阐述了人ASCs-EVs促进再生膀胱血管化的具体信号通路机制；最终证实人ASCs-EVs结合水凝胶复合支架成功支持膀胱扩大再生，为突破临床膀胱扩大术瓶颈确立了新型治疗手段。