囊性纤维化动物模型 z-bo 2020-12-07 282

　　Background: Animal models of cystic fibrosis (CF) are the key to understanding the pathogenesis of CF and developing treatment strategies. Considering that CF affects multiple organs including the gastrointestinal tract, lungs, pancreas, liver, and reproductive organs, it is necessary to produce animal models that accurately capture all disease aspects. In the past 20 years, in vivo CF-related research has mainly used transgenic mouse models. These mouse models have been shown to be very valuable for studying the pathophysiology of CF, but they have limitations. The obvious differences between mouse and human anatomy and physiology mean that the phenotypes observed in humans are not always replicated in mice. Studying certain characteristics of CF airway disease in mouse models has proven to be problematic. Because CF mice cannot develop features including mucus obstruction, chronic bacterial infection and persistent inflammation. Among all clinical manifestations related to human CF, progressive lung disease is the main cause of morbidity and death in patients. Therefore, accurate animal models of the occurrence and development of CF lung disease are essential for studying pathogenesis, identifying potential therapeutic targets and testing experimental treatments. With the establishment of CF mouse models, it is important to consider species characteristics when developing animal models of CF lung disease. Features that may affect species selection include airway cell structure, especially the distribution and abundance of submucosal glands, the advantages of alternative chlorine secretion pathways, and the protection of the structure and function of cystic fibrosis transmembrane conduction (CFTR). Since the development of the CF mouse model, advances in genetic engineering have facilitated the generation of several alternative models, including CFTR knockout rats, knockout ferrets, two knockout pig models, and pigs containing the common PHE508DEL CFTR mutation. CFTR knockout and PHE508DEL mutant CF rabbit models have also been reported early. Due to the similarity between sheep and human lungs, a CF sheep model has been proposed for a long time. Although the CF sheep model has not yet been established, the rapid development of gene editing can promote its production in the near future. This review details the phenotypes of airway diseases in existing CF animal models and examines their advantages and disadvantages.

　　Pathophysiology of lung disease: Before studying the phenotypes observed in animal models, it is necessary to understand the underlying pathophysiology of CF lung disease. Under normal conditions, the CFTR protein acts as an epithelial anion channel, responsible for cyclic AMP-dependent chloride and bicarbonate secretion, as well as the regulation of epithelial cell sodium channels (ENaC). When the CFTR gene is mutated, CF is produced, which subsequently disrupts epithelial ion transport. To date, more than 2000 pathogenic CFTR mutations have been identified. These mutations are classified into 6 categories according to the mechanism of CFTR dysfunction, including defective protein synthesis and processing, dysfunctional channel regulation and transmission, and reduced protein synthesis and stability. The most common CF-causing mutation is the class II mutation PHE508DEL, and about 90% of CF patients carry a copy. Although the role of CFTR in transepithelial ion transport is widely accepted, the exact mechanism of the development of CF lung disease has long been debated. Several hypotheses have been proposed regarding the pathogenesis of CF lung disease. There is a lot of evidence to support the theory called the "low volume" hypothesis. This theory assumes that the decreased transmembrane chloride transport due to dysfunctional CFTR, and the increased sodium absorption due to the lack of CFTR-dependent inhibition of eNAC, creates an imbalance of ions, resulting in water absorption that penetrates into the tissues, and reduces the airway surface layer. (ASL) Hydration. Depleted ASL causes dehydration of the mucus layer, and sticky mucus adheres to the airway surface. Mucus stasis and impaired mucociliary clearance (MCC) lead to ineffective clearance of inhaled microorganisms. Bacteria adhere to the airway mucus and eventually form a biofilm, effectively avoiding antibacterial substances and host neutrophils. Over time, more resistant and atypical organisms have gathered in the respiratory tract, including mycobacteria, yeasts and fungi. The state of chronic lung infection causes a continuous inflammatory response, leading to airway tissue destruction, bronchiectasis, gradual loss of lung function, and ultimately respiratory failure.

　　CF mouse lung disease phenotype research: The disease phenotype and severity of the CF mouse model tends to be heterogeneous due to the diversity of genetic background and the differences in gene targeting strategies used to produce animals. CF mice did not show any lung pathology, and new evidence suggests that they do exhibit some human lung disease phenotypes. Similar to the early lung phenotype of human CF airways, multiple studies have shown that CF mice show altered respiratory mechanics. When the lungs of CF and wild-type mice are repeatedly exposed to inflammatory stimuli (lipopolysaccharide of Pseudomonas aeruginosa), they will cause inflammation. The lungs of CF mice undergo remodeling and morphological changes, while the wild-type The airway of mice can be effectively restored. Recent studies have also shown that CF mice spontaneously develop Bodella respiratory tract infections. Although CF mice exhibit the characteristics of lung disease, their use has certain limitations because they do not show the severity of human CF-pulmonary disease consisting of chronic respiratory infection, inflammation, mucus blockage, and progressive bronchiectasis. Pathological characteristics. Some theories can explain why CF mice cannot develop the overt lung disease observed in humans. One is to up-regulate modified genes in CF mice, leading to partial correction of partial ion transport defects. Advantages of calcium-activated chloride channel (CaCc) secretion pathway in airway epithelium of CF mice. This adaptive mechanism compensates for the deficiency of CFTR-mediated chloride ion transport, thereby correcting the potential ion imbalance and protecting the mouse airway from disease. Except for the upregulation of CaCc in the airway of CF mice, the role of cAMP-mediated CFTR pathway in mouse airway epithelium is usually not dominant. The lower respiratory tract of adult CF mice does not show electrical defects that are characteristic of human CF airways, including reduced cAMP-mediated chloride secretion and high sodium absorption. The overactivity of ENaC plays an important role in the pathogenesis of CF lung disease because it is believed to contribute to the failure of ASL and subsequent events, including mucus accumulation, damaged MCC, infection and inflammation, and lung tissue damage. The lack of high sodium absorption in the lower airways of the CF mouse model may explain the lack of lung disease development. The structure and function of CFTR in mice are different. Studies on the homology of CFTR show that mouse CFTR is only 88% conserved with human CFTR at the amino acid level. Therefore, mouse CFTR channels exhibit different pharmacology to human CFTR. Human airway CFTR protein is mainly expressed in ciliated epithelium and submucosal glands, so the histological differences between mice and airway can explain the occurrence of lung disease in CF mice. Mice and human airways are different in cell structure; the distal airways of humans are mainly composed of ciliated cells, while the lower airways of mice are mainly non-ciliated, secretory, rod-shaped cells. In addition, the human airway has many submucosal glands in the trachea and bronchus, while the mouse airway has only a few glands in the larynx and proximal trachea. Since submucosal glands are implicated in human CF airway disease, their scarcity in the distal airway of mice may lead to a lack of lung pathophysiology. Environmental factors and host-pathogen interactions may also play a role. CF lung disease cannot be effectively modeled in the absence of pathogens, so CF mice can prevent the development of lung diseases that are commonly observed in CF under semi-sterile and usually SPF conditions. However, this theory does not seem to be proven because CF mice raised in a non-sterile environment still cannot develop lung disease.

　　CF mouse upper airway and trachea phenotype: Some CF mouse strains show mild phenotypes in the upper airway and trachea. The observed features include dilated submucosal mucosal glands in the nasal cavity, atrophy of serous glands in the sinuses, goblet cell hyperplasia of the nasal septum, and a decrease in ASL of nasal epithelial cells. The CFTR//- mouse model also showed significant tracheal abnormalities, such as incomplete cartilage rings and decreased smooth muscle area. Damage to the MCC of the trachea of the CFTRtm1HGU and CFTRtm1UNC strains has also been reported. However, these findings have been found by others, and MCC is not affected in CFTRtm1UNC mice. The nasal epithelium of CF mice exhibited bioelectrical abnormalities similar to those of human CF airways, including reduced chloride transport and high sodium absorption. These deficiencies have been well documented using nasal potential difference (NPD) measurement technology. An evaluation involves placing a fluid-filled cannula electrode (connected to a millivoltmeter) on the nasal lining to measure the electrical potential generated by the transmission of ions through different salt solutions across the epithelium. All CF mice exhibit reduced cAMP-mediated secretion of nasal epithelial cells, which is characteristic of human CF airways. The activity of Enac is present in the human CFN nasal epithelium, and is also observed in the nasal epithelium of most CF mouse models, including knockout, CFTR, F508 and CFTRG51D strains. When amiloride (a drug that blocks Enac-mediated sodium absorption) is perfused through the nasal epithelium of CF mice, a significant depolarization reaction occurs, which is consistent with the presence of high sodium absorption. Since the nasal epithelium of CF mice accurately reflects the electrophysiological characteristics of human CF airways, it has been proven to be useful for detecting therapeutic agents that replace dysfunctional CFTR (ie gene therapy) and restore normal ion transport. Mouse strains carrying PHE508DEL and GYL51ASP mutations are particularly useful for detecting compounds that are CFTR processing and trafficking (CFTR correction) and defective channel gating (CFTR enhancer). However, the enhancement of human CFTR by some CFTR enhancers has no effect on mouse CFTR, most likely due to differences in the properties of mouse CFTR channels. Once developed, the HCFTR CF mouse model will facilitate the detection of small molecule drugs.

　　Homologous CFTR knockout mouse model: It is speculated that the generation of a CF mouse model under a mixed genetic background leads to up-regulation of modified genes, thereby preventing the development of lung diseases. To test this theory, the CFTRtm1UNC-CF mouse strain was re-developed under a single genetic background and was called B6CFTRtm1UNC. Interestingly, B6 CFTRtm1UNC mice showed signs of lung disease, including bronchiole mucus retention, tissue fibrosis, hyperinflated alveoli and alveolar wall thickening. The lungs of B6 CFTRtm1UNC mice also showed recruitment of inflammatory cells, and an influx of neutrophils was observed. This inflammatory disease phenotype appears to be spontaneous, because the analyzed mice were placed in SPF conditions and no respiratory pathogens were detected before inflammation or the onset of inflammation. The nasal PD display indicates that the same CF mice exhibit reduced chloride secretion. However, unlike the mixed background CF mouse strain, the response to amiloride does not seem to be different between the same species of CF mouse and the wild type, suggesting that the homologous CF mouse does not exhibit high sodium absorption in the nasal epithelium. . B6 CFTRtm1UNC mice challenged with P. aeruginosa showed reduced ability to control infection. It is speculated that the homologous B6CFTRtm1UNC mice develop lung disease due to the lack or failure to activate the alternate chloride ion conduction pathway that is usually present in the mixed background CF mouse model. The B6 CFTRtm1UNC mouse is one of the few models of inflammatory lung disease. Therefore, it has been used to explore the long-standing debate about whether CF airway inflammation is spontaneous or caused by infection. Although B6 CFTRtm1UNC mice showed aspects of CF lung disease, the original mixed background CF mouse strain was used.

　　Β-ENaC mouse model: In existing mouse models, the difficulty of re-understanding the pathophysiology of CF lung disease has led to the development of β-EnaC mice (also known as SCNN1B mice). β-Enac mouse is a transgenic mouse model, which can overexpress the β subunit of eNAC in the lung, thereby mimicking the abnormal sodium ion transport in the human CF airway. β-Enac mouse airway remodels the key process of the pathogenesis of CF lung disease, increasing airway sodium absorption, leading to ASL exhaustion and insufficient mucus clearance. Therefore, β-Enac mice exhibited CF-like lung disease characterized by high mucus secretion, mucus obstruction of the conductive airways, MCC damage (directly measured by microdialysis), goblet cell metaplasia, and neutrophil gas. Tract inflammation. Preliminary studies using adult β-Enac mice failed to detect spontaneous bacterial infections in the airways. However, when challenged intratracheally with Haemophilus influenzae and P. aeruginosa, adult β-eNac mice, it was compared with wild type Compared with mice, the ability to remove pathogens is impaired. Recent studies on the longitudinal analysis of BAL have shown that neonatal β-Enac mice have spontaneous lung infections. The same study also found that the bacterial burden and proportion of the lungs of β-Enac mice after infection decreased with age, which was attributed to the maturation of the immune system that occurred after birth. β-eNac mice provide a relevant model for studying the pathogenesis of CF lung disease, especially the interaction between ion transport, ASL and MCC. Pulmonary diseases simulated by β-eNac mice have been confirmed for the development of new respiratory diagnostic tools to locate and measure the heterogeneity of lung diseases. In addition, β-Enac mice can be used to evaluate a range of treatments, including treatments for ASL exhaustion, mucus obstruction, and inflammation. For example, β-eNac mice can be used to test eNAC inhibition to restore normal sodium transport. These methods include the use of eNAC antagonists, the use of short interfering RNAs (siRNA) to silence eNAC expression, and inhibit the proteases that activate Enac. However, because the expression and function of CFTR in the β-ENaC model have not changed, it is not suitable for the treatment of replacing, correcting or enhancing CFTR.

　　CF rat model: Compared with larger animals, rats have the advantages of short pregnancy (21-23 days) and early sexual maturity (8 weeks), allowing rapid reproduction. Compared with large animals, rats have less animal feeding costs and are widely used for research purposes, which means they have good characteristics in physiology, pharmacology and toxicology. Unlike mice, rats have extensive submucosal glands throughout the cartilaginous airways (trachea and primary bronchi), similar to humans.

　　CFTR gene knockout rat airway disease: CFTR-/-rat summarizes the important characteristics of CF. lung disease observed in humans. However, the cells tend to show expansion due to increased mucus levels within the cells, indicating defective mucus secretion. Morphologically, 6-week-old CF rats have abnormal tracheal development. Compared with wild-type rats, the area of tracheal cartilage and glands is reduced. CF rats also showed significant depletion of ASL and significantly reduced the depth of ciliary fluid (PCL). Preliminary results indicate that young CF rats exhibit a highly acidic airway surface, similar to CF pigs. A recent study showed that the ASL pH of children with CF did not decrease. Despite the depletion of ASL, the mucociliary transport (MCT) of young CF rats does not appear to be affected (<3 months). However, early evidence suggests that with the age of the animal and the development of airway submucosal glands, the MCT rate is significantly reduced compared to wild type. Preliminary studies have also shown that at 6 months of age, the mucus of the airway submucosal glands of CF rats is blocked. Not present in the airway of CF rats In spontaneous infection and inflammation, there was no significant difference between wild-type and CFTR genotypes. Although spontaneous infection does not exist, new evidence shows that it is similar to CF mice. The ability of CF rats to clear Pseudomonas aeruginosa infection is weakened.

　　CF ferret model: Due to the biological and anatomical similarities between ferrets and human lung cells, normal ferret lungs have been used to simulate human lung infections, including severe acute respiratory syndrome and influenza viruses. Like humans, ferret airways have submucosal glands throughout the trachea and primary bronchus, which express high levels of CFTR. In addition, the main secretory cell type in ferrets and human proximal cartilage airways is goblet cells. Ferret CFTR and human CFTR (95%) also have a high degree of amino acid sequence protection. Therefore, the bioelectric and pharmacological properties of CFTR are Ferrets are similar to humans.

　　 CFTR knockout ferret model airway disease research: CF ferret shows severe lung phenotype. Mucus blocks the small and large airways and, in some cases, leads to complete blockage. In addition to the presence of mucus, there are also trapped air and deflated alveoli (atelectasis). Dilation of submucosal glands and ducts, goblet cell hyperplasia, and the presence of inflammatory cells and bacterial colonies in the mucus were observed in the airways of CF ferrets. Bronchopneumonia, necrosis, inflammation and lung carbuncle are present. Occasionally, CF ferrets also experience sinus mucus accumulation and inflammation. When the migration of fluorescent beads in the peripheral trachea was used to measure MCC, CF ferrets showed a significant reduction.

　　CF pig model: The similarities between humans and pigs in anatomy, physiology, biochemistry, size, lifespan and genetics make pigs a suitable candidate for simulating human diseases. Pig lungs and human lungs have a variety of anatomical and histological features, including similar tracheal-bronchial tree structure and the abundance of airway submucosal glands. Pigs have previously been used to simulate lung diseases, including inflammation and infections such as chronic bronchitis. As human CF lung disease progresses in the lifespan of individuals, the lifespan of pigs allows long-term studies on the pathogenesis and therapeutic evaluation of lung disease, which is impossible in rodent or ferret models because their lifespans are relatively long. Shorter or worse. The amino acid sequence of porcine CFTR is also 95% conserved with humans. The electrophysiological characteristics of porcine respiratory epithelium and submucosal glands are similar to those observed in humans. Airway disease in CF pig model: CFTR-/-, CFTRΔF508/ΔF508 and CFTR-/ΔF508 pigs spontaneously show the main characteristics of human CF lung disease within a few months after birth. These manifestations include airway inflammation, infection, and tissue weight. Plastic, mucus accumulation and obstruction. CF pigs showed heterogeneous airway remodeling, some cases showed goblet cell hyperplasia and airway wall thickening. CF pigs with airway obstruction, including atelectasis, hyperinflation, air traps, and pneumonia. The purulent substance appears to block the trachea and bronchi and usually contains bacteria, neutrophils and macrophages. Conclusion: CF mouse models have been used to study many aspects of CF, but their application in airway-related research is limited because they only show the mild features of CF lung disease, using bacterial challenge to induce airway disease Has limitations. Although the nasal epithelium of CF mice provides a platform for exploring pathogenesis and evaluating treatment, the lack of obvious lower respiratory disease manifestations and the high phenotypic heterogeneity between strains means that new models are needed. β-eNac mice provide a useful alternative to CF mice because it mimics lower airway obstruction, although it is not suitable for all applications because the expression and function of CFTR are not affected. Efforts to overcome the shortcomings of existing mouse models have led to the development of new CF animal models, including CF rats, ferrets, and pig models. CFTR knockout rats showed many lung phenotypes, including airway surface defects, CF-like electrical distribution in the nasal cavity and tracheal tissue, abnormal mucus production, and tracheal malformations. The lung diseases modeled by CF ferrets have many similarities with those observed in humans. Simultaneous airway inflammation, infection, and mucus obstruction provide a very valuable platform for further elucidating the pathogenesis of lung diseases and the treatment methods in the lung environment closely replicated with human CF patients. Pigs and humans share multiple airway characteristics, making them suitable for the development of CF lung disease models. The recently produced CFTR knockout and PHE508DEL mutant pig models exhibit features of lung disease, including inflammation, airway obstruction and infection, which will help basic and preclinical airway related research. Animal models that accurately summarize the characteristics of human CF-pulmonary disease will provide researchers with resources for experimental lung therapy, identify new therapeutic targets, and clarify the complex mechanisms of CF lung disease. Although a perfect model of CF lung disease does not exist, each animal model has unique advantages and can be used complementarily to study CF-related issues.