| BackgroudOnychomycosis is the most common infection of the nails (more than50%),which indicates infection by dermatophytes (tinea unguium), yeasts, andnon-dermatophyte moulds. The incidence of onychomycosis has been increasing inrecent years. Some of the contributing factors causing this disease are humidity,occlusive footwear, repeated nail trauma, and concurrent disease, such as diabetes,poor peripheral circulation, and HIV infection, as well as other forms ofimmunosuppression. The incidence increases with age, and nearly30%of patientsare older than60years. Although infrequent, this infection can affect children.Over the past few years, in vitro experimental onychomycosis models have beenacting as a valid instrument for the preliminary determination of the fungal ability inpenetrating the nails. Nails clipped from the hoof plates of healthy volunteers andanimals have been used in these models. For better performance and observation,hoof plates from animals such as porcine are more popular than that from humandimension-wise.The establishment of a scientific, economic, simple in vitro onychomycosismodel for monitoring onycomycosis pathogenicity and evaluation of antifungalactivity has a great significance.Infection caused by dermatophytes are widespread and increasing in prevalenceon a global scale so that it has been considered a major public health concern insome areas of the world. Among dermatophytes, the anthrophilic fungusTrichophyton rubrum represents the most clinically important species, whichaccounts for as many as69.5%of all dermatophytoses in humans. T. rubrum is themost common pathogen caused the dermatophytosis of nail and skin stratumcorneum in human and animals. Moreover, T. rubrum infections are often intractableand relapses.The prevalence of T. rubrum infections and the anthrophilic nature of thisspecies make it a good model for the study of pathogenic superficial fungi. In recentyears, many researches have been focus on the biological characteristics of T.rubrum, but there are still many issues need to be explained, especially on the relationship between T. rubrum and keratin, the growth in keratin medium and theimpact factors of pigment. Currently, there is still no relevant experimental studiesand reports on the biological characteristics of T. rubrum.One of the remarkable properties of dermatophyte is its ability to decomposekeratin. Keratins are a group of fibrous proteins, which is the structural componentof vertebrates skin and nails, contains a cystine-rich matrix with disulfide bonds tomaintain high stability. T. rubrum can parasitize on stratum corneum, hairs and nailplates. T. rubrum–secreted proteases are considered to be important virulence factorssince they can digest the keratinized tissues and collagen protein, elastin and otherproteins into short peptides and amino acids for the further digestion and facilitatethe invasion. Among them, keratinase is the major enzyme involved in thepathogensis process. There have been many studies on keratinase production fromdifferent species of dermatophytes. However, we still know nothing about how T.rubrum could adapt and responds to the adverse environments and how to regulatetheir gene expression.Therefore, this study attempts to elucidate the pathogenesis of onychomycosisand the related virulence factors. Our research includes the following three parts:Part1A preliminary study on an in vitro onychomycosis modelObjective To observe the different growth of the fungi in the hoof plateMethod we established an in vitro model of onychomycosis using porcine hoofplates. An “O” ring was developed to seal the fungi on the surface of hoof plate andfungal suspension was applied to the “O” ring; The dynamic penetration of hoofplates was observed daily during the incubation process. Observing the growth oflocalized fungi on the hoof plate with histopathological examination and scanningelectron microscope.Result Our experimental onychomycosis model succeeds in encouraging T.mentagrophytes and T. rubrum invade and to penetrate the porcine hoof plates, butCandida albicans can not.Conclusion Our experimental onychomycosis model succeeds in mimicking allinfectious stages of the fungal invasion. The model allows us to campare the abilityof the fungal to penetrating the nails. Part2The influence of different nutrition on the T. rubrum growthin vitroObjective To investigate the influence of different nutrition on the T.rubrum growthin vitro.Method T. rubrum suspension was cultureed on a different nutrient agar plate, suchas potato dextrose medium, Sabouraud medium, trypsin glucose medium, keratinglucose medium, glucose medium and blank medium at30℃. Investigating thecolony morphology, color, sporulation and measure colony diameter for1month.Result From the results of morphology and size of colony, both potato dextrose andSabouraud medium provide rich nutrition for the growth of T. rubrum. However,keratin medium is relatively poor nutritive so that the colony size of T. rubrum issmaller than that of PDA and SDA. Glucose, the only carbon and nitrogen source,can’t provide enough nutrition for T. rubrum growth.Conclusion The growth of Trichophyton rubrum in the medium with protein arebetter than that without protein. The PH regulates the conidial pigmentation.Part3Investigation of the keratinase activities of onychomycosispathogenObjective Compare different substrate for keratinase activity of T. rubrum andobserve pathogenicity in adverse enviroment.Method Using different substrate (keratin, glucose, trypsin) to induce T. rubrum tosecrete keratinase. Measure the keratinase activity of T. rubrum after damaged byoxidants, itraconazole, terbinafine under different substrate.Result Strains grown on keratin medium showed high activities than those grown ontrypsin medium. Under some oxidative damaging effect, T. rubrum keratinaseactivity increased with keratin as substrate.Conclusion Activity of keratinase produced by T. rubrum is correlated to the typesof the substrates. T. rubrum can regulate the expression of keratinase against oxidantdamage under keratin substrate. |
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