RESUMO
Keloid is a major type of skin fibrotic disease, with one prominent feature of extensive accumulation of extracellular matrix (ECM) components, and another feature of pain/itching, which is closely related to the peripheral nervous system (PNS). However, the molecular pathogenesis of these two prominent features still needs to be further explored. In the present study, we performed single-cell RNA sequencing (scRNA-seq) on clinical earlobe keloid samples and adjacent normal skin samples and constructed a keloid atlas of 31,379 cells. All cells were clustered into 13 major cell types using cell-type-specific markers. Among them, fibroblast, vascular endothelial cells, and smooth muscle cells were defined as the ECM-related populations according to their ECM-associated functions. Also, we found that Schwann cells (SCs) were the main neuron cells of PNS in the skin. Interestingly, the cell proportions of ECM-related populations, as well as SC were increased significantly in the earlobe keloid compared to the adjacent normal tissues, suggesting an important role of these cell types in the development of the earlobe keloid. Comprehensive cell-cell interaction analysis at the single-cell level revealed a strong interaction between SC and ECM-related subgroups which might be mediated by SEMA3C signaling pathways and MK/PTN gene family, which are found to be mainly involved in promoting cell proliferation and migration. Moreover, further exploration of the interactions of ECM-related populations and SC in different keloids, including earlobe keloid, back keloid, and chest keloid revealed an increasing amount of TGFß-TGFß receptor interactions in chest/back keloids as compared to earlobe keloid, which suggested the anatomic site-specific pathogenesis in different keloids. Altogether, these findings suggested the interactions between ECM-related populations and SC contributing to the earlobe keloid formation and helped us to better understand the pathogenesis of keloids.
RESUMO
OBJECTIVE: To investigate the relationship between bacterial biofilm and acute otitis media by observing the feature of bacterial biofilm formation in middle-ear mucosa in the rat model of acute otitis media and to study the possibility of application this rat model in bacterial biofilm research. METHODS: A total of 30 healthy, male SD rats were studied, 24 animals served as experimental group were bilaterally injected with 50 µl of Streptococcus pneumoniae suspension (1 × 10(8) CFU/ml) via a transbullar approach into the middle ear cavity after anesthesia and six animals were bilaterally inoculated equivalent saline account for control group. At day 1, 3, 5, 7, 10 and 14 after inoculation, bilateral middle-ear mucosal specimens were collected from three infected animals and one control animal for scanning electron microscopy (SME). Membranoid substance attached the bilateral middle ear mucosa were collected under the microscope from the other one infected animals, which were prepared for confocal laser scanning microscope (CLSM) with immunofluorescence in situ labeling technique and light microscopy using Gram staining. RESULTS: At the early stage of infection (1 day, 3 days), lots of bacterial adhesion, permanent planting in the local regions of the middle ear cavity and microcolonies formation were found, with mixed phagocytic cells, showing a primary bacterial biofilms formation. In the middle term of infection (5 days, 7 days), mature bacterial biofilm scattered on the mucosal surface, formed characteristic three-dimensional structure of "mushroom-shaped" towers. At the late inflammatory period (10 days, 14 days), the bacterial biofilms presented signs of recession. CLSM with FITC-ConA and PI double staining in situ labeling and light microscopy using Gram staining indicated that bacteria and polysaccharide matrix within the biofilms were viable. CONCLUSIONS: These preliminary findings provide evidence that bacterial biofilms form at the early phase of acute middle ear infection and it may be an important factor in the development of recurrent or persistent otitis media. The rat model of AOM established in this study may be an ideal animal model facilitating the bacterial biofilms research.