iPSC诱导分化及植入脱细胞基质构建生物人工肺脏研究

Bio-artificial lung has become a new direction of the end-stage lung disease treatment. Adult stem cells have limited sources and poor proliferative capacity, and embryonic stem cells are faced with many ethical and rejection problems. As for this, both are not suitable as seed cells. IPSC, derived from an reprogrammed somatic cell, which has multiple differentiation potential and can avoid the defects of adult and embryonic stem cells, is an ideal seed cell. But preparation of iPSCs in a large scale is more difficult due to its low efficiency of differentiation. Preliminary studies have successfully induced iPSC to differentiate into AT2 by using ActivinA and SAGM phased induction system. The aim of this study was to explore the mechanism by which Integrin signaling pathway can induce iPSC to differentiate into AT2 through tracking iPSC's induction and differentiation process and thus efficiently induce iPSC to differentiate into alveolar epithelial cell. The improved decellularization technology by whole lung perfusion langendorff in preliminary studies can prepare well decellularized scaffolds; bioreactor has been tested and meet the design requirements. The research may provide a new theoretical basis and therapeutic direction for patients with end-stage lung diseases through seeding AT2 derived from iPSC and endothelial cells in the lung acellular matrix scaffold, culturing in a bioreactor, and then examining the performance of tissue engineered lung and evaluating the lung orthotopic transplantation in vivo.

构建生物人工肺已成为终末期肺病治疗的新方向，成体干细胞来源受限且增殖能力差，胚胎干细胞存在伦理及排异问题，作为种子细胞均不适合。iPSC源于体细胞重编程而具有多向分化潜能，避免了成体和胚胎干细胞的缺陷，是较理想的种子细胞。但iPSC分化效率低，大量制备较为困难，前期研究采用ActivinA 和SAGM分阶段诱导体系，已成功诱导iPSC向AT2分化，本研究拟通过追踪iPS细胞诱导分化全过程，探索Integrin信号途径调控iPSC向AT2分化的机制，从而高效诱导iPSC向肺泡上皮细胞分化。前期研究改良的langendorff全肺灌注脱细胞技术可制备良好的脱细胞支架;生物反应器经测试达到设计要求。拟通过在肺脏脱细胞基质支架种植iPSC来源AT2和内皮细胞，在生物反应器培养，对构建的组织工程肺进行性能检测和原位肺移植在体评价，为终末期肺病患者应用组织工程人工肺替代治疗提供新的理论依据和治疗方向。

肺移植是终末期肺病患者唯一有效的治疗方式，生物人工肺为肺移植带来了新方向。构建生物人工肺的三个关键要素包括：脱细胞肺支架、种子细胞和生物反应器。本研究中，为了制备出理想的脱细胞肺支架，我们改良了langendorff全肺灌注脱细胞技术，应用一种新型去垢剂:十二烷基醚硫酸钠 (SLES)用于脱细胞，对比传统去垢剂脱氧胆酸钠(SDC)和十二烷基硫酸钠(SDS)，进行了一系列对比评价：组织学染色、免疫荧光、DNA 扫描电镜、免疫印迹等检测，SLES制备的肺支架三维结构和细胞外基质蛋白保留更加完整，显示出更好的效果。iPSC源于体细胞重编程而具有多向分化潜能，避免了成体和胚胎干细胞的缺陷，是较理想的种子细胞。本研究首次采用人尿液细胞重编程获得iPSC，iPSC再诱导分化为肺泡II型上皮细胞，并表达肺泡II型上皮细胞的表面活性蛋白，方法更为简便有效，具有无创性，为临床研究和应用带来更大便利。生物反应器通过模拟肺脏的生理状态，使种子细胞更加有效地到达肺远端区域。本研究通过在脱细胞肺支架种植人肺II型上皮细胞，在自制的生物反应器内培养，更好的实现了脱细胞肺支架的细胞化，为后续生物人工肺的细胞培养打下了基础。最后，通过脱细胞肺支架同种异体皮下移植6周，来验证制备的人工肺在体内的融合情况、降解情况、周围免疫排斥情况、细胞浸润情况，为人工肺的临床应用奠定了理论基础。. 本研究阐明了构建生物人工肺较为理想的脱细胞方法，找到了一种较为理想的去垢剂，制备出了较为理想的脱细胞肺支架；首次探索了人尿液细胞来源的iPSC作为理想的种子细胞，制备了较为满意的生物反应器；异体动物实验取得了较为满意的结果。总之，本研究具有较大的科学意义和临床价值。