Screen Differentially Expressed Genes In Skin Of Mice After Intradermal Inoculation Of Prototheca

Part I:Establishment of cutaneous protothecosis model in miceObjective:To develop a mouse model of cutaneous protothecosis.Methods:A total of48BALB/C mice were equally divided into4groups, including group A(immuno-suppressed mice inoculated with P.zopfii var.portoricensis suspension of1×109cfu conidia ml-1),group B(immuno-suppressed mice inoculated with suspension of1×106cfu conidia ml"1),group C(healthy mice inoculated with suspension of1×109cfu conidia ml"1),and group D(healthy mice inoculated with sodium chloride solution). Mice were treated with subcutaneously injected P.zopfii var.portoricensis suspension or sodium chloride solution on the abdomen skin. On day7th,14th and28th investigation of abdomen skin appearance change in each group were recorded, in addition, pathological and mycological examinations were also performed.Results:In group A, B and C, papule and abscess of inoculation sites were noted in all mice, demonstrating100%morbidity, besides, ulceration and crusts were also noted. The diameters of the skin lesions in group A, B and C on day7th were all significantly larger than those on day14th and28th (P<0.05), were(6.75±1.09)mm、(5.88±1.17)mm and (5.96+0.99)mm. However, no significant difference was found among the three groups on day7th and14th (P>0.05),group A was (4.38±0.86) mm for maximum on day28th.Pathology revealed necrosis, abscess and granuloma formation. Multiple spores were observed by HE and PAS stain as well as direct microscopy of pus. Cultures of tissue samples grew Prototheca. The mice in group D remained uninfected.Conclusion:Mouse model for Protothecosis may be established by subcutaneous inoculation of Prototheca suspension of healthy or immuno-suppressed mice. Part II:Screen differentially expressed genes in skin of mice after intradermal inoculation of ProtothecaObjective:To screen differentially expressed genes in skin of mice after intradermal inoculation of Prototheca by using cDNA microarray and discuss the potential profunction of these genes in order to have updated knowledge of cutaneous protothecosis.Methods:The total RNA of skin tissues of immuno-suppressed mice inoculated with Prototheca and corresponding normal skin tissues were isolated and labeled by reverse transcription reaction for cDNA.Labeled cDNA were hybridized with cDNA microarray.After scanning and image processing,the different gene expression profiling was investigated. Part of differentially expressed genes were chosen to detect by real-time quantitative RT-PCR.Results:Totally9902genes had more than two fold changed expression in skin of mice after intradermal inoculation of Prototheca,which include4974up regulated and4928down regulated genes. Bioinformatics analysis revealed that function of some differentially expressed genes involved inflammatory response,immune system process,signal transduction, cell adhesion and so on. Some interesting pathways such as chemokine signaling pathway, Jak-STAT signaling pathway and Toll-like receptor signaling pathway were found.The results of real-time quantitative RT-PCR exhibited a trend toward the microarray data.Conclusion:The development of cutaneous protothecosis involved the change of many genes.Genes differentially expressed in skin of mice after intradermal inoculation of Prototheca indentified by using cDNA microarray may be benefit to the study of pathogenesis and therapy of cutaneous protothecosis.