人成体肺干细胞在脱细胞肺支架中区域特异性分化及调控机制研究

Adult stem cells are responsible for the cellular homoestasis and renewal in their homed organ. The differentiation of adult stem cells is determined, therefore they are the ideal seeding cells in organ engineering. Recently, human lung stem cells (hLSC) are found in adult human lung. We isolated hLSC, studied its differentiation in porcine decellularized lung scallfod (DCLS), and found that hLSC showed region-specific differentiation in porcine DCLS, that is to say that hLSC differentiated into airway and alveolar epithelial cells and pulmonary vascular cells respectively when seeding into the airway and vessel of DCLS. We hypothesis that the region-specific distribution of the residual signal molecules in the DCLS matrix and the different activation of lung development-associated pathways may mediate the region-specific differentiation of hLSC in DCLS. In this project, firstly we will make clear the region-specific differentiation of hLSC in DCLS. Then we will compare the components of the different parts of DCLS matrix, the activated lung development-associated pathways in the process of region-specific differentiation of hLSC in DCLS, and primarily identify the possible signal molecules and pathways involved in the regulation of region-specific differentiation of hLSC. Finally we will identify the mechanism of the region-specific differentiation of hLSC in DCLS by exploring the differentiation of hLSC in DCLS in the condition of intervening these molecules and pathways. The results from this project will give us new knowledge on the differentiation of hLSC in three-dimensional DCLS, and new basis for the making of bioengineering lung using hLSC and DCLS.

成体干细胞的分化方向已被限定为所居器官组分细胞，是器官工程的理想种子细胞。新发现人肺脏存在成体肺干细胞(hLSC)。我们在分离培养hLSC基础上发现：hLSC在脱细胞肺支架(DCLS)可发生区域特异性分化，即植入DCLS气道和血管的hLSC分别分化为气道/肺泡上皮及血管内皮/平滑肌细胞。故推测：DCLS基质中残留分化诱导信号分子的区域特异性分布及不同肺发育相关信号通路活化可能是hLSC区域特异性分化的重要机制。本课题拟通过比较DCLS不同区域基质成分差异、hLSC区域特异性分化过程中肺发育相关信号通路活化特征，初步筛选调控hLSC区域特异性分化的可能基质分子和信号通路；通过观察在干预所筛基质分子和信号通路条件下hLSC在DCLS中分化改变，明确hLSC在DCLS区域特异性分化调控机制。研究结果可为hLSC在三维DCLS中的分化调控提供新认识，为基于hLSC和DCLS构建人工肺提供新依据。

肺移植是终末期肺病唯一有效治疗手段，供体肺缺乏严重制约肺移植的开展，组织工程肺是解决供体肺来源的可行途径，种子细胞、支架材料和种子细胞在支架材料中生物行为调控则是组织工程肺的主要瓶颈问题，脱细胞肺支架和成体肺干细胞是组织工程肺很有前途的支架材料和种子细胞。前期参考文献分离培养了成体肺干细胞(LSC)并发现LSC在脱细胞肺支架（DCLS）中有区域特异性分化的倾向，本项目拟深入研究LSC在DCLS中的区域特异性分化行为及可能的调控机制。主要进行了下述实验：（1）收集肺切除手术患者标本，选取病变旁正常肺组织，采用酶消化法分离、培养人成体LSC（krt5+p63+细胞），采用类似方法从小鼠肺组织中分离培养了小鼠远端气道LSC，建立了从人或鼠肺组织标本中分离培养LSC方法；（2）建立、优化了灌流法制备大鼠DCLS的方案，进行了胶原蛋白、糖蛋白等信号分子的切片组化染色；（3）制备人iPSCs并诱导分化为LSC，建立了稳定的iPSCs制备和LSC定向诱导分化方法，采用对外周血单核细胞进行重编程技术（仙台病毒感染转入重编程因子Oct4、Sox2、Klf4、cMyc）制备了人iPSCs，通过激活肺发育相关的信号通路和分子，模拟肺发育过程（定型内胚层、前肠内胚层、腹侧化前肠内胚层和LSC），初步将iPSCs成功诱导分化为LSC（NKX2.1+），诱导的LSC能进一步分化为肺泡上皮；（4）观察了krt5+p63+小鼠肺干细胞对博来霉素引起的小鼠肺纤维化的治疗作用，发现krt5+p63+小鼠肺干细胞可以明显减轻博来霉素导致的肺纤维化，其作用于促进肺脏再生和抑制肺纤维生成有关；（5）进行了“全自动器官脱细胞支架制备仪”的研制，已完成原型样机制备和专利申报。上述iPSC分化肺干细胞和灌流法制备DCLS方案的建立和优化，为进一步研究肺干细胞特别是iPSC分化肺干细胞在DCLS中的生物学行为、组织工程肺脏的人工制造以及肺干细胞药物的开发奠定了基础。