| Fungal infection is one of the most common disease in department of dermatology, about50%of the clinical cases. Recently, the incidence of fungal infection was increasing year by year with the abuse of antibiotics and immunosuppressive agents, the increasing patients of tumor、AIDS、organ transplantation, and the deterioration of environment. Serious environment has become a crucial factor for the denelopment and aggravation of various disease. As the destruction of ozonosphere, ultraviolet exposed to the earth is increasing, which intensified the hazard of ultraviolet.The impact of UV on a variety of diseases such as bacteria, viruses, cancer et al. has largely been reported, but the effect of UV on fungal infections is still indefinite.UV in the natural environment includ UVA (320to400nm), UVB (280to320nm) and UVC (<280nm),of which UVA and UVB take a large part, and a small number of research reports of the UVC. The impact of UVA and UVB on the skin included the ability to cause erythema, pigmentation, damage to physical structure and local immune et al.. However the distinction of UVAand UVB to induce the clinical manifestations and mechanisms of the skin photodamage has not yet been completely described. More than400kinds of pathogenic fungi has been found in the environment, which is one of the most common pathogenic microorganisms around us. Fungi are heterotrophic eukaryotes, which can be divided into the yeast-like fungi and mycelium-like fungi by traditional morphology. Most fungi are common in the environment, prodicient at sensing its surroundings and react in order to promote their survival in the environment, which allowed them able to interact with plants, animals or people in a variety of maners to form a symbiotic state, latent infection or pathogenic state.In addition to the role of fungal own virulent factors, the host defense system will also affect the development, turnover and prognosis of fungal infections. Skin barrier function can be divided into two kinds:the generalize skin barrier and skin barrier of a narrow points.The former refers to the summation of the physical barrier, as well as immune barrier of the skin, pigment barrier, and nerve barrier and other skin functions related. The other refers to integrity of the skin, especially the"brik-wall structure" consisted of the stratum corneum and the lipids. Skin barrier structure is based primarily on the stratum corneum, epidermis lipids and natural moisturizing factor, as well as various epidermal proteins and the metabolism of inorganic salts. Epidermal lipids consist of ceramides, free cholesterol and free fatty acid. The lipid content from the basal layer of the epidermis to the stratum corneum was gradually increased, which was discharged from cell gap to the stratum corneum,constructing a barrier to prevent moisture loss. The natural moisturizing factor was decomposed from the filaggrin in the epidermis, containing amino acids, pyrrolidone carboxylic acid, Urocanic acid, lactic acid, urea and other substances of low molecular weight which combines with water to maintain the skin barrier function. Immune barrier function included immunity of cell level involving of T cells, Langerhans cells, phagocytic cells, mast cells, neutrophils et al, as well as molecular level immunity involving a variety of cytokines, tumor necrosis factor, Urocanic acid, anti-microbial peptide-based.So far, a variety of study has been reported about the effect of UV on the skin barrier, and the development of secondary skin infection with various bacteria, viruses and skin cancer. However few studies dad shed light on the fungal infention after UV radiation. And the majority of the experimente focus on singal UVA or UVB for their mechanism of action, ignoring the combined effects of UVA and UVB in the environment. Therefore, we use the SUV-1000solar simulator as a light source, and selected guinea pigs as the our experimental animal after preliminary experiments, and choosed the most common pro-animal pathogenic fungi Trichophyton mentagrophytes as the experimental strain to build skin sunburn model after different dose of ultraviolet radiation.And we detected the skin barrier in order to study the mechanism of skin photodamage caused by solar ultraviolet radiation. Meanwhile Trichophyton mentagrophytes infection model after radiation was contructed in order to have a further understanding of the effect and mechanism of ultraviolet radiation on fungal infections.Chapter one:Determination of minimal erythema dose of the experimental animals1. Objective:To detect the minimum erythema dose of C57BL/6, ICR mice and guinea pigs and determine which is the optimal experimental animals.2. Methods:We used SUV-1000sunlight simulator as light source, and the MED determination was divided into dosese in descending order of eight irradiation hole. UVA and UVB output power was measured before each irradiation. We had10guinea pigs, each for a group of eight spot dose. We had32C57BL/6and ICR mice were respectively, and every four mice shared a group of spot dose irradiation. Skin of the experimental animals were prepared before irradiation fixed under SUV-1000solar simulator, and the skin was attached close to the irradiation hole. At the same time, the eyes were masked to avoided radiation. Doses of UVA radiation for Guinea pig ranged from160to2500mJ/cm2, and UVB about15to200mJ/cm2with an increment by approximately1.414times. Doses of radiation for C57BL/6and ICR mice was UVA160to2500mJ/cm2and UVB10to150mJ/cm2. Reaction of skin erythema in each group were observed within48hours after irradiation. The minimal erythema dose was evaluated by the appearance of visible skin erythema24hours after irradiation. The average of minimal erythema dose of three experimental animals was calculated as the final minimal erythema dose for three kinds of mice respectively.3.Results:The minimal erythema dose of three mice after simulated sunlight irradiation were calculated respectively. UVA498mJ/cm2, UVB42mJ/cm2and the total dosage of540mJ/cm2for C57BL/6mice; UVA644.9mJ/cm2, UVB55.4mJ/cm2, and the total dosage of700.3mJ/cm2for ICR mice; UVA1430mJ/cm2, UVB122mJ/cm2, and the total dosage of1552mJ/cm2for guinea pigs. Considering the operation of simulated sunlight, skin preparation of experimental animal, and the operation of fungal infection, we select guinea pigs as the optimal experimental animals for our experiment.Chapter two:Detecting the skin barrier function of guinea pigs after solar-simulated radiation and analyzing the relationship between the skin barrier and fungal infection.1. Objective:To construct animal model of sunburn after radiation of simulated sunlight,and to construct animal model of Trichophyton mentagrophytes infection after radiation. Then investigating the connection between the irradiation of simulated sunlight and the damage of skin barrier and the infection of skin with Trichophyton mentagrophytes.2Methods:(1) fungal infection after simulated sunlight irradiationWith exposuing the back skin of guinea pig to SUV-1000solar-simulated ultraviolet(SSUV),we determined the average minimal erythema dose(MED) for them.40guinea pigs were divided into5groups of which the first1,2,3,4group were respectively irradiated with OMED、0.5MED、1MED、4MED of SSUV for three consecutive days. The next day after irradiation, pre-prepared Trichophyton mentagrophytes suspension was applied evenly on the irradiated parts of the guinea pig back skin The5th group, as blank control group, didn’t receive radiation after hair removal, using physiological saline insteated of Trichophyton mentagrophytes suspension as an untreated blank group. Guinea pigs of every group were placed in a singal cage respectively,air-dried and feeded alone to observe changes of the clinical presentation in the guinea pig back skin since the date of vaccination for a period of28days. The clinical presentation of erythema, scaling, crusting, erosion is counted as1point respectively. The score has to be added for a total when a variety of clinical performance occered.(2) detection of the skin barrier after infection6geniea pigs were randomly selected from each group the next day after exposure. We take skin with the skin drills at the irradiated part after anesthesiaed with10%urethane,and put it into10%paraformaldehyde. for HEstaining, and CD4, CD8antibody staining. At the same time, the inguinal lymph nodes, as skin draining lymph nodes of the guinea pig back skin was handled as before. (3) identification of fungal infectionDirectmicroscopy, culture, and histopathologic methods were adopted to verify the fungal infections. Direct microscopic examination and culture of fungus was taken at4days after inoculation. We scraped the dander and hair of guinea pig back skin, under a microscope with10%potassium hydroxide for conventional microscopic examination, and leaving a small amount of dander and hair to inoculate on the SDA medium containing chloramphenicol and cycloheximide every four days, till28days after infection to observe the skin and hair fungal infection.7to10days after culture, we observe the colony morphology, and picked up a little culture colonies with a sterile inoculating needle to staine with lactic acid phenol Medan under the microscope for identification.10days after inoculation, two guinea pigs were randomly selected from each group to be killed, to The whole skin organization was incised from the site of infection for PAS stain and further identification of the infection results.(4) statistical analysisDatas were analysised by SPSS13.0. Scores of group1、2、3、4after infection were presented with x±s, of which scores among groups were analyzed through repeated measure ANOVA,but insteated by Greenhouse-Geisser test if datas were not satisfied with Mauchy’s test of sphericity. And scores between groups were analyzed through LSD test. Datas of area under the curve, time of the first infection and time of the hightest score were presented in x±s manner,and analyzed by one-way ANOVA. P<0.05was defined as to have statistical significance.3. Results:General change after infection:Group5,as blank control group,did not have the appearance of fungal infection. As to the erythema of skin inflammatory reaction, group3and4occurred at3days after inoculation, the2th group began at4days after inoculation,while group1developed the appearance of skin erythema reaction5days after inoculation. During infection, the clinical performance of Group4day was more severe compared with group1,2,3of the same.21days after infection, skin lesion of guinea pigs of group1and2restored graduall, with the evidence of defluxing crust, leaving slight erythema and a small amount of desquamation on the back skin. Skin lesions of groups3and4began to heal23days after infection. Score of skin lesions were statistically analyzed, showing that the difference among4groups was statistically significant (p=0.049). Score of skin lesions of Group4had a statistically significant difference with group1and2(P-value was0.022,0.037respectively), In contrast, difference between group4and group3was no significant (P>0.05), For the1,2,3th group,there exhibited no significant difference between one and the other as well.(P of the1、2group was0.747,1、3group was0.377,2、3was0.503). The difference of area under the curve among4groups has no statistically significance (P=0.070),the same as the difference of the first infection time (P=0.066) and time of the hightest score(P=0.729).The results of skin barrier function test showed that the physical barrier was damaged after the irradiation, showing a increased tendency with the increase of irradiation dose. Immunohistochemical staining of CD4, CD8T cells showed that CD4and a small amount of CD8can be seen in the skin and lymph nodes of the lth group. While CD4of group2,3,4decreased in the skin and lymph node incontract with group1, and CD8increased. We found no significant difference among groupl,2,3,4for HE staining of Inguinal lymph nodes.Fungus examination showed that guinea pigs of1,2,3,4th group were all successes in fungal infection. Group5, as blank control group, was failed in fungal infection. We found hyphae and spores full of visual field under microscope in the skin dander of guinea pigs of1,2,3,4th group4days after inoculated with Trichophyton mentagrophytes. Fungus in the culture was confirmed Trichophyton mentagrophytes by observing the colony morphology and lactic acid phenol Medan staining. After PAS staining of guinea pig skin tissue sections of group1to4,we found a large number of spores and a small amount of mycelium distribution by microscope dyed dark red in the stratum corneum, the hair root and hair shaft, but no hyphae and spores in Group5.Conclusions of full text:1. Experimrnt of minimum erythema dose of Guinea pigs, of C57BL/6mice and ICR mice show that the guinea pig is most suitable animal for fungal infection experiment after simulated sunlight radiation,with the advantages of broad back skin sutable for eight-hole spot exposure in one guinea pig to minimize the error; and it was feasible to be depilated with barium sulfide,which could have less damage to the skin; and the guinea pigs were completely docile to be exposed under simulated sunlight; finally, the broad back skin were more easily to be inoculated with the fungus suspension and facilitate to observe the clinical manifestations of the infection.2Skin erythema could be evoked by simulated sunlight after exposure to more than1×MED UV dose, but radiation lower than1MED dose could not induce the skin sunburn reaction of guinea pigs. The skin barrier was injured obviously after irradiation of SSUV, which dad a aggravated tendency with the increase of UV dose. Simulated sunlight irradiation did not have any influence on the incidence of fungal infections, which was proved by our datas that positive rate of fungal infection for irradiated group and non-irradiated group were equally100%. But a significant effect can be seen of dosage of simulated sunlight on the severity and self-healing of skin lesions after fungal infections, which was supported by the performance that with the increase of dosage of radiation, the skin lesion was more severe and need more time to recover to normal.According to results above,we can get the conclusion that skin barrier damaged after solar-simulated radiation may be the root of different degree of severity of fungal infection, however the concrete mechanism between was still unknown,which need further research. |
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