| Popis: |
Recent legislation has accentuated the importance of developing novel in vitro toxicology testing strategies which avoid the use of animals. The focus of this study was to use in vitro epithelial models to characterize the point of change between protective and deleterious responses in the lung following exposure to well-characterized commercial polymers. The first step was to develop and characterize the Normal Human Bronchial Epithelial (NHBE) primary cell model, which resulted in an organotypic, multi-differentiated bronchial model which was then used to elucidate the phenotypic and genotypic responses to polymers. The NHBE model exhibited evidence of ciliogenesis and mucus production comparable to native human bronchial epithelium. Conventional toxicology techniques were utilized to establish the doses required to induce sub-toxic responses in the model a lower (TD05) dose to induce a protective response and a higher (TD20) dose to induce a more injurious response. Transmission (TEM) and Scanning Electron Microscopy (SEM) were employed to examine changes in the inter-cellular morphology and revealed the TD05 dose led to regional hypertrophy but that the TD20 dose resulted in cellular degradation and general degeneration of tissue integrity. Transcriptomic tools (GeneSpring, Metacore) were applied to microarray data to compare gene expression profiles across NHBE cultures (multiple donors) and with a commercial tracheal model (EpiAirway, single donor), as well as to characterize the molecular pathways implicated in response to each dose. This demonstrated that the TD05 dose induced alterations of genes and pathways representative of protective and inflammatory responses but the TD20 dose induced airway injury, wounding and remodeling. It was also found that although the nature of responses were similar, the EpiAirway tissues were over-sensitive and presented a shorter viable experimental window, therefore the NHBE was considered the optimal in vitro model to assess toxicological responses of the pulmonary epithelium. |
