Study Of Chitin Synthnase Gene Expression In Onychomycosis

Objective:Onychomycosis is one of the most common nail disorders and caused by dermatophytes, molds and yeasts. It is difficult to eradicate with drug treatment.There is not fully clear that the pathogenesis of onychomycosis at present. Chitin that is in the fungal cell walls of which biosynthesis is a complicated process, which the key enzyme is chitin synthnase(CHS). There are more than one categories of CHS isozyme in different fungi. The isozymes play an important role in maintaining cell wall integrity, normal hyphae growth and asexual spores and so on. There are also found that some lack of CHS gene in the pathogenic fungi will weak the growth of strain and infect it’s ability at the same time. Chitin does not exist in animals and plants, so CHS is considered to be the ideal target of antifungal drugs. This study is to select the hyphae of the main invading pathogen, Trichophyton rubrum,are ofter interwoven with the nail plate to determine a simple in vitro onychomycosis model.This article focused on the difference of CHS expression in onychomycosis during different time using real-time fluorescent quantitative PCR technique and to explore the role of CHS in fungal infection the porcine hoof and to study CHS of Trichophyton rubrum as effective antifungal drug targets.Method:1 Adopting porcine hoof infected by Trichophyton rubrum to determine a simple in vitro onychomycosis model.2 Applying real-time fluorescent quantitation PCR technology to establish chitin synthase (CHS2 mRNA) standard product and its standard curve.3 Detecting the alternations of chitin synthase (CHS1 mRNA and CHS2 mRNA) in Onychomycosis in lth,2th,3th,4th,5th weeks after infected by Trichophyton rubrum with the standard products as established previously.Result:1 Establishing Onychomycosis in vitro model by adopting porcine hoof infected by Trichophyton rubrum is simple and economically viable.2 The standard products and standard curve were constructed by real-time fluorescent quantitation PCR technology.Using cDNA as the standard product,which is the products of reverse transcription of total RNA,we got better amplification curve,standard curve,and melt curve.The standard curve demonstrates well linear relationship (house-keeping gene GAPDH:R2=0.999,goal gene CHS2:R2=0.999, R2>0.98) and accurate quota can be achieved in a broad scope.The amplification rate is satisfactory (house-keeping gene GAPDH:E=89.18%,goal gene CHS2:E=96.08%,0.8<E<1.2).3 Compared with the control group, There was no obvious change of CHS1 mRNA expression in the experimental group after seven days (P>0.05), but there was significantly increased after 14 days (P<0.05), while the expression of CHS2 mRNA at all time points increased significantly (P<0.05).Conclusion:1 Our experimental onychomycosis model succeeds in mimicking all infectious stages of the fungal invasion, and observation decks change.It is economically feasible for assessing the role of pathogenic fungi on porcine hoof.2 The standard curve between the goal gene CHS2 and the housekeeping gene GAPDH established by the relative quota method presented good linear relationship and repeatability.3 There is a significant differencein the gene expression of chitin synthase in the experimental group and the control group (P<0.05),which indicating that chitin synthase may plays an important role in the process of Trichophyton rubrum infection porcine hoof.