| Trichophyton mentagrophytes is a common pro-human and animal dermatophytes, not only can cause skin, nails and other parts of the superficial infections, but can also cause kerion, abscesses and granuloma of the deep infections . deep infection of skin caused by Trichophyton mentagrophytes is relatively rare, but the trend is increasing year by year, and difficult to cure, easy to relapse and re-infection. Current, There were some research on the pathogenesis of superficial infection caused by Trichophyton mentagrophytes , most studies focused on the transcriptional level, while little studied on deep infection caused by Trichophyton mentagrophytes in the pathogenesis.Skin is composed of epidermis, dermis and subcutaneous tissue,the epidermis is divided into stratum corneum, transparent layer, granular layer, spinous layer, basal layer, the dermis is divided into papillary layer and reticular layer. Constituted mainly by cells in the epidermis, dermis mainly composed of fibrous connective tissue, bone collagen and elastic fibers. superficial infection of skin caused by Trichophyton mentagrophytes confined to the epidermis, while deep infection of skin caused by Trichophyton mentagrophytes had penetrated the epidermis to the dermis and subcutaneous tissue. whether existence of different virulence factors on the differences of infected sites and the severity caused by the same kind of fungi, pending further studies.This paper choose Trichophyton mentagrophytes is the research object, to study the differences between granuloma strains and tinea strains from biological characteristics-genotype-the level of protein,trying to find a more powerful virulence factors and to guide clinical treatment. Objective: To screen differentially expressed proteins from granuloma strains and tinea strains and explore the pathogenesis of the deep and superficial infection. Method:1. plate culture and small steel ring culture method On 4 granuloma strains and 4 tinea strains in 25℃and 37℃respectively , to observe the colony morphology and microscopic morphology.2. Guinea pig infection experiments: Preparation bacterial suspension of 4 granuloma strains and 4 tinea strains. glucocorticoid immunization intervention, inoculate the same amount of bacterial suspension by the skin surface and subcutaneous inoculation methods,observe lesions. Direct microscopic examination,fungal culture and histopathology results of skin lesions verify infection. Normal guinea pig skin as a negative control.3. PCR on the NTS region and its three sub-areas Tmis0, Tmis1, Tmis2 Region of 13 granuloma strains and tinea strains.4. Proteomics: Select 1 granuloma strains and 1 tinea strains, proceed two-dimensional electrophoresis, Coomassie brilliant blue staining, PDquest software analysis, pull relevant differences points in protein gels, MS detection, acquisition protein information, identified and analysis through NCBI database.Results:1. Colony morphology: 37℃, 4 strains of granuloma were better than 4 strains of tinea in growth; 27℃,no significant difference between the two strains in growth; at 37℃better than 27℃in growth of 4 strains of granuloma; at 27℃better than 37℃in growth of 4 strains of tinea (see Figure 1).2. Microscopic morphology: 37℃: 4 strains of granuloma mycelium complete and full, more small conidia, and a few large conidia, 4 strains of tinea mycelial dry shrinkage strain occurs, and a fracture phenomenon, 27℃: of difference is not obvious between 4 strains of granuloma and 4 strains of tinea (see Figure 2).3. Guinea pig infection model:3.1 Surface inoculation: 4 strains of granuloma: the next day, erythema appeared in the skin, gradually expanding, two weeks after the erythema surface covered with a thick layer of scales, 3 weeks after erosion, broken collapse, a fracture surface of the hair were found in two strains; 4 strains of tinea: the first 4 days appeared light erythema, then the surface covered with thin scaly skin lesions, no erosion, ulceration. 4 weeks after Granuloma strains and tinea strains gradually becoming better.3.2 intradermal vaccination: granuloma strains: the first 5 days , appear nodules, and gradually increased, 2 weeks the size of a fingernail. While tinea strains no nodules appear.3.3 Histopathology: 3 weeks after, granulomatous nodules for pathological, PAS staining showed: granulomatous inflammation, a large number of macrophages, few neutrophils, lymphocytes and plasma cells (see Figure 4a), bead-like spores and septate hyphae in subcutaneous tissue, (see Figure 4b,c).3.4 Fungal microscopy and culture: cut tissue culture, see a fluffy colonies after 2 weeks (see Figure 5a,b), picked mycelium see a large number of myceliums microscopic colonies(see Figure 5c).4. PCR amplification: 3 sub-regions were shown with a different types(see Figure 6), which TmiS0 divided into 4 types, TmiS1 divided into 5 types, TmiS2 divided into 2 types.5. Protein two-dimensional electrophoresis and PDquest software analysis: measured different protein points between granuloma strain and tinea strain are more than 100 proteins, the height expression points of granuloma strain about 62. 5 Meaningful protein: enolase, heat shock protein 60 and 70 and small heat shock protein, serine/threonine protease, glutamate carboxypeptidase and a number of hypothetical proteins.Conclusion:1. There are differences in biological characteristics between Granul -omatous strains and tinea strains, granulomatous strains more resistant to temperature.2. The pathogenicity to guinea pigs of Granuloma strains and tinea strains is different , granuloma strains susceptible the host than tinea strains , and the notable inflammatory reaction.3. There is no difference in genotype strains between granuloma strains and tinea strains.4. obtained meaningful proteins: enolase, heat shock protein 60 and 70 and small heat shock protein, serine/threonine protease, glutamate carboxypeptidase and a number of hypothetical proteins.5. there are some differences between granuloma strains and tinea strains about biological characteristics and functions of proteins, but no significant difference between genotypes. |
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