基于干细胞肺内移植及基因编辑方法建立人类遗传性呼吸窘迫综合征模型

As the economy and society development, environmental pollution and heredity caused lung diseases are increasing recent years, but the treatments of different types of lung diseases are still challenging, the mortality is still not under control. Therefore development of the diagnosis and treatment of lung disease is imminent. In this project, we use in vitro lung stem cell culture, combined with combined CRISPR/Cas9 technology to induce site-specific mutation of surfactant protein B. Through the flow cytometry to screen single GFP expressed lung stem cell clone. The 121 ins2 site and ATG site of SPB will be chosen for the induced mutation, finally two SPB gene mutated lung stem cell clones will be obtained and for transplantation. Then the mutated stem cells were implanted to Nod-Scid mouse lung through transplantation to make a human-mouse chimera lung. This chimera lung can be used as a hereditary lung disease model for scientific research and clinical treatment of related lung diseased, even for the drug screening.

肺表面活性物质是一种调节气－液交换过程中肺泡表面张力的脂蛋白。 SFTPB (surfactant B，SPB) 缺陷会引起新生儿急性呼吸窘迫综合征，并进一步导致慢性呼吸综合症等疾病。 本项目结合体外肺脏干细胞培养，利用CRISPR／Cas9技术将携带有SPB基因特定序列的引导 RNA 和表达绿色荧光的Cas9质粒在肺干细胞中共转染，然后通过流式细胞方法筛选表达绿色荧光单细胞克隆，构建SPB突变的肺脏干细胞。选择SPB基因外显子的121 ins2位点以及基因的起始密码子位置作为突变的靶点，最终期望得到2个人肺干细胞SPB基因突变克隆。体外移植SPB突变的肺脏干细胞细胞到 Nod-Scid 小鼠肺脏，构建人－鼠嵌合的遗传性肺病模型。并进一步利用此肺病模型进行常用肺部疾病用药处理，如pirfenidone 等，寻找合适的治疗药物及用药组合，服务于科学研究以及临床治疗。

严重肺部感染是对人类健康的一大威胁，伴随而来的是高昂的医疗费用、长期的住院需求和高死亡率。为应对抗生素耐药性的出现和持续的细菌感染，我们迫切需要一种新的抗菌策略。天然抗菌肽LL-37，有助于清除临床肺部感染的主要病原体——铜绿假单胞菌(Pseudomonas aeruginosa, PAO1)。通过基因工程技术，将LL-37基因导入成年小鼠远端气道干细胞(DASCs)，并将编辑后的细胞原位移植到损伤的小鼠肺灶中。移植后的细胞大规模整合并长期释放宿主防御肽，保护小鼠免于细菌性肺炎和低氧血症。此外，在成人支气管中分离、扩增的DASC利用相同技术进行基因编辑，并成功构建人工肺。我们基于干细胞的基因传递治疗平台提出了一种新的策略，用于处理具有未来转译机会的复发性肺部感染。