雪貂囊性肺纤维化模型的建立与分析

Cystic pulmonary fibrosis (CPF) is the most common life-threatening autosomal recessive condition among people of mixed European descent. CPF is caused by mutations in an epithelial chloride channel encoded by Cystic fibrosis transmembrane conductance regulator (CFTR) gene. CFRD is associated with worsening clinical status, including reduced pulmonary function, increased frequency of pulmonary exacerbations, and a decline in nutritional status. Furthermore, CFRD leads to increased mortality compared with CPF patients without diabetes. Tissues affected in CPF include the lung, pancreas, liver, intestine, gallbladder, sweat gland, and male reproductive tract. CPF lung disease is characterized by a chronic and non-resolving activation of the innate immune system with excessive release of chemokines/cytokines into the airways. Chronic infection and impaired immune response eventually lead to pulmonary damage characterized by bronchiectasis, emphysema, and lung fibrosis. Early diagnosis and treatment are vital to improving clinical outcome of CFRD patients. Therefore, there is a strong rationale for the identification of mechanisms and pathways underlying the exaggerated inflammatory response in CPF lung disease. The lack of an animal model for CPF-related diabetes (CFRD) has made it difficult to dissect how the onset of pancreatic pathology influences the emergence of CFRD. We evaluated the structure and function of the neonatal CPF endocrine pancreas using a Cftr-knockout ferret model. Although much has been learned through this CPF ferret model, limitations in the ability of this model to recapitulate spontaneous lung disease seen in CPF humans have created a need for additional efforts on which to study CPF. To this end, in this project, we plan to generate and characterize a lung-specific Cftr-knockout ferret CPF models induced by intranasal Cftr RNAi. This new specifically animal models closely resemble human CPF disease seen in newborns and conveniently characterize their adult phenotypes, and may help elucidate pathophysiologic mechanisms of CPF lung disease and lead to new therapeutic approaches.

囊性肺纤维化（Cystic pulmonary fibrosis，CPF）是一种具有家族常染色体隐性遗传性的先天性疾病，极易发生呼吸道反复感染而严重损害肺功能，多数病人仅能成活数年到十余年，死亡率极高。CPF 的发生与跨膜调节因子基因Cftr突变，导致绿脓杆菌感染、气道黏液梗阻和进行性肺组织坏死有关，其发病机理和治疗策略有待深入研究。目前，在活体水平上尚无合适手段来研究CPF的发病机制、治疗策略和预后评估；临床上患者对治疗反应存在很大差异，至今尚无明确的治疗靶点和合理的治疗方案。雪貂由于具有与人类类似的粘膜下腺结构和Cftr相似性，而被认为适合用于CPF的研究。鉴于此，本研究拟在以往全身性敲除Cftr基础上，利用慢病毒滴鼻方式，首次构建雪貂肺部特异性Cftr敲除模型，探索其生物学行为，包括肺部Cftr敲除雪貂的建立、动态病理变化和肺部功能分析，为研究CPF发病机制和治疗方案提供实验科学依据

雪貂的呼吸系统细胞生物学特性（黏膜下腺和CFTR）与人类具有相似性，是一个适合研究CPF的模型动物。虽然全身性敲除CFTR的雪貂已经出生并且表现出一系列与人类似的全身性各种器官的临床病理特征，但是人类CPF最终致命的器官是肺部纤维化，所以制备一只在肺部特异性CFTR敲除的雪貂来进行肺部功能、动态病理变化的分析显得尤为重要。本课题你拟在以往全身性敲除CFTR的基础上，利用克隆或CRISPR/Cas9介导下构建雪貂肺部特异性CFTR敲除模型，探索其生物学行为，包括肺部Cftr敲除雪貂的建立、动态病理变化和肺部功能分析，为研究CPF发病机制和治疗方案提供实验科学依据。研究结果表明，1）首次建立一个养殖雪貂的实验动物房，需要具备如下条件：通风、恒温、特制的饲养笼具、特制的高蛋白饲料。2）pU6-floxedRFP-shCFTR载体构建后转染到新生或21天胎儿成纤维细胞的效率非常低。3）首次尝试使用CRISPR介导的ctRNP+ssDNA显微注射受精卵，胚胎移植后没有得到阳性雪貂。4）首次研究了卵母细胞体外成熟时间为24h、二氧化碳培养箱温度38.5℃最佳，TCM-199培养基中添加激素有助于提高成熟率。5）雪貂卵母细胞GV染色质构型可以作为判定卵质量的标准。6）首次研究了雪貂卵巢体外最佳的保存时间和温度分别为12小时和15-20℃。7）受精卵收集的方式：交配后43-45小时从输卵管伞向子宫方向冲洗。8）公貂输精管结扎手术部位是在阴囊开口，可以精准定位输精管。9）雪貂受精卵显微注射之前离心力的最低阈值为4500g。10）原核注射后过夜培养，胚胎移植后出生率要比胞质注射低，但是阳性率比胞质注射高。