Gene Expression Of JAK/STAT Pathway In The Patients With Psoriasis

BackgroundPsoriasis is a common chronic inflammatory disease, its morbidity is1～4%. Clinically, psoriasis is divided into4types:psoriasis vulgaris, psoriasis pustulose, psoriasis arthropathica and psoriasis erythrodermicum, in which psoriasis vulgaris is the most common type, its clinical features include erythema, papule and silver scale. Apart from skin lesion, there may be metabolic syndrome, such as hyperhomocysteinemia, hyperlipidaemia, atherosclerosis, obesity and diabetes mellitus, etc. So psoriasis can bring about lots of pain in body and psychology to the patients, meanwhile psoriasis often costs the patients a lot because of its recurrence. Therefore, to study the pathogenesis of psoriasis is meaningful.The main pathology of psoriasis are abnormal proliferation of keratinocytes, parakeratosis, angiogenesis and inflammatory cells infiltration. Its exact etiology and pathogenesis remain unclear. Recently, the studies showed that psoriasis is a disease of multifactorial inheritance with the interaction of genetic and environmental factors, lots of studies indicated that immune factors including immune cell, cytokines involve in the pathogenesis of psoriasis. Therefore, it is worthy to investigate the role of immune factors in the pathogenesis of psoriasis. According to recent researches,40kinds of hormones, cytokines, growth factors and bioactivator involve in regulating the proliferation and differentiation of keratinocytes, so do some kinds of signal transduction system.Most of researchers believe that psoriasis is a T cell mediated autoimmune disease. Studies showed that there are a series of cytokines secreted by activated T cell in the lession of psoriasis, such as IFN-γ、IL-1、IL-6、IL-8and TNF, etc. Those cytokines can combine relative receptors of target cell and then induce the change of expression of keratin in keratinocytes via JAK/STAT signal transduction system. JAK/STAT signal transduction system seems to involve in the pathogenesis of psoriasis, however, the exact Transduction mechanisms of STAT is unclear yet.JAK(Janus kinase)/STAT(signal transducer and activator of transcription, STAT) is a very important signal transduction pathway of cytokines. After activated by the combination of cytokines and their relative receptors, JAK/STAT signal transduction pathway can pass on the signals from cell membrane to cytoplasm and to cell nucleus eventually, then induces some sorts of biological effects which involve in physiological and pathological reaction of human. Nowadays the genes and protein of STAT of mammal have been isolated and cloned, there are7kinds of STAT: STAT1,STAT2, STAT3, STAT4, STAT5a, STAT5b and STAT6.Rresearches indicated that abnormal activated STAT1and STAT3can promote the proliferation and differentiation of cells and suppress the apoptosis of cells which lead to occuring of some diseases.SOCS (Suppressor of cytokine signaling) was discovered in1997, which is an important negative agent of JAK/STAT signal transduction pathway. SOCS1and SOCS3are the most important ones of those agents. The mechanism of negative regulation of JAK/STAT pathway of SOCS include:suppressing the activity of kinases by combining JAK, suppressing the activity of STAT by suppressing the combination of STAT and their relative receptors, mediating the degradation process of proteasome which is signal protein dependent.Psoriasis is a disease of immune disorder, abnormally activated T cell can secrect a series of cytokines, which can induce the change of expression of keratin in keratinocytes, and the relationship of IFN-y and K17’expression is clear, IFN-γ can activate STAT1which combine to the K17gene promoter, then the expression of K17is induce in the keratinocytes above the basal layer of epidermis. Others factors involve in the process through the same signal transduction. Cytokines’ receptors passing on signals via JAK is necessary because it is short of tyrosine kinase itself. JAK activates STAT and then affects genetic transcription. Based on those relative research results, it is worthy to study the role of JAK/STAT signal transduction pathway in the pathogenesis of psoriasis.Objective1to collect the tissue of lesional and non-lesional area of10psoriasis patients, then isolate keratinocytes from those tissue.2The expression levels of STAT1, STAT3, SOCS1, SOCS3genes were valuated by using real-time quantitative PCR technique.3Relationships of the expression levels of STAT1, STAT3, SOCS1, SOCS3mRNA with the pathogenesis of psoriasis were analyzed, then deduce the role of JAK/STAT signal transduction pathway and SOCS in the pathogenesis of psoriasis.4Discussing the relationship of STAT and SOCS to study the pathogenesis of psoriasis deeply.Methods1Object of study:Patients with psoriasis have typical skin lession and the pathological diagnosis, clinical manifestations of progressive stage include:new rash appears constantly, old rash expanding increasingly, there are red plaque and thin scales, surrounded by inflammatory flush, itch is significant; there are no history of using glucocorticoid, immunosuppressants, vitamin A acid drugs and other treatments within1month before collecting sample, the patients have no other system diseases. a total of10patients were picked according to the above standard.2Collecting a part of tissue from lesional area and non-lesional area of the above patients, then remove subcutaneous tissue individually, leaving epidermal layer and dermis.3Specimens were cut to8mmx3mm size and washed three times by PBS, then digest the specimens one night using1.6U/mL dispase in4℃. On the second day, the keratinocytes were isolated and saved in the cryopreserved tubes to extract RNA.4Extract total RNA from the specimens, then detect the OD260/280value of RNA using ultraviolet spectrophotometer to determine the purity of RNA; take a suitable amount of RNA in1%agarose gel electrophoresis and1x TAE Electrophoresis liquid and observe RNA electrophoresis image using gel imaging analyzer, if there are two clear28S and18S stripe, suggests that RNA samples’ quality is good. Store those RNA in-80℃freezer according to the above standard.5The design and synthesis of primer:retrieve NCBI GenBank database, primers was designed respectively according to different exons, GAPDH is designed as an internal control. Design STAT1and STAT3, SOCS1and SOCS3and GAPDH PCR primers by using Primer Premier5software.6Synthesize cDNA by reverse transcription7Real-time PCR:the volume of PCR reaction system is20μL, including cDNA2.0μL, dH2O6.4μL, SYBR(?) Premix Ex TaqTM (2×)10.0μL,10μmol/L upstream and downstream primers0.8μL individually. Real-time PCR was conducted in CFX96fluorescence quantitative PCR. Each gene of each specimen repeat three holes. Using three-step method to amplify. The average Ct value of each gene of each specimen was calculated, the Ct value of STAT1and STAT3, SOCS1and SOCS3minus the GAPDH Ct as delta Ct values.8Data analyses were conducted by using SPSS13.0statistics software, the comparison between two variables was conducted by homogeneity of variance analysis and t test, the relationship between two variables was conducted by Pearson correlation analysis. P<0.05means difference is statistically significant.Results1The expression levels of STAT1, STAT3, SOCS1, SOCS3mRNA(△Ct) in the keratinocytes of lesional area(-1.9±1.6.0.6±1.6、0.1±1.0、-0.6±1.1) significantly increased than those observed in non-lesional area(-1.9±1.6、0.6±1.6、0.1±1.0、-0.6±1.1).2The expression levels of STAT1, STAT3mRNA of keratinocytes were found to positively correlate with the expression levels of SOCS1and SOCS3mRNA individually.Conclusion1The expression levels of STAT1and STAT3mRNA in the keratinocytes of lesional area significantly increased than those observed in non-lesional area, which indicate that JAK/STAT signal transduction pathway might involve in the pathogenesis of psoriasis. It is regarded that those cytokines secreted by Th1cell combine to the relative receptors in the keratinocytes, then JAK is activated and STAT is phosphorylated, phosphorylated STAT can recognize and combine to the special response elements of DNA of target genes and regulate the transcription of target genes, thus the formation of psoriasis lession is promoted.2The expression levels of SOCS1and SOCS3mRNA in the keratinocytes of lesional area significantly increased than those observed in non-lesional area, and the expression levels of STAT1, STAT3mRNA of keratinocytes were found to positively correlate with the expression levels of SOCS1and SOCS3mRNA individually. It is regarded that cytokines activate JAK/STAT signal transduciton pathway and promote the formation of psoriasis lession, at the same time, SOCS1and SOCS3were induced to suppress the JAK/STAT pathway, in this way can SOCS supress the pathogenesis of psoriasis.