The Establishment Of An In Vitro Model Of Psoriasis In Serum-free William's Medium Supplemented With Psoriatic T Cells And Streptococcal Antigens

Objective: To investigate the in vitro method of psoriatic skin culture and to establish the living organism model of psoriasis. The aim of the study is, by culturing psoriatic organ, we are targeting to discover the etiological factors and pathogenesis of psoriasis. Furthermore, the ideal model can be used for precaution, drug screening and clinical therapy of psoriasis. The experiment can also detect the expression of keratin and cytokine in psoriatic skin and establish special markers of psoriasis; analyze the roles which T cell, streptococcal antigen, keratin 17, interferon-gamma, tumor necrosis factor-alpha and interleukin-8 play in pathogenesis of psoriasis.Methods: A facility was devised for culturing the psoriatic skins. According to psoriatic pathogenesis, psoriatic lesions were cultured at the air/liquid interface in serum-free William's medium supplemented with psoriatic T cells and streptococcal antigens. Psoriatic characterizations were compared between un-cultured and 6-day-cultured lesions by macros- copic observation, light microscope and electron microscope. Furthermore, keratin 17 expressed in psoriatic skin and normal control group was dete- cted by immunohistochemistry method and western blotting; the expres- sions of interferon-gamma, tumor necrosis factor-alpha and interleukin-8 were detected by enzyme-linked immunosorbent assay within different cases and groups. All the data were analyzed by SPSS and EXCEL stati- stically.Results: (1) Macroscopic observation, light microscope and electron microscope were used to observe the structure and characterization of skin organs. Psoriatic lesions which cultured at the air/liquid interface in serum-free William's medium supplemented with psoriatic T cells and streptococcal antigens after 6 days still maintained the psoriatic structure and characterization ; On the contrary, psoriatic lesions which cultured in pure serum-free William's medium went different from psoriatic chara- cteristics; Normal skins which cultured at the air/liquid interface in serum-free William's medium supplemented with psoriatic T cells and streptococcal antigens after 6 days turned to be psoriatic. (2)The results of immunohistochemistry method: High expression of K17 was noted in the cultured and uncultured psoriatic lesions but no expression in normal skin; There was no significant difference of K17 between 6-day-cultured and uncultured lesions (P>0.05), while obviously significant difference with normal ones in statistics (P<0.01). (3)The results of SDS-PAGE and western-blotting: molecular weigh of Keratin 17 is about 46kDa according to low molecular weight protein marker; Keratin 17 band appeared in 6-day-cultured and uncultured psoriatic lesions. There is no K17 band in normal skins. (4)The results of enzyme-linked immunosorbent assay: different psoriatic cases cultured in three different conditions over expressed interferon-gamma, tumor necrosis factor-alpha and interleukin-8. In contract, normal skin group can hardly express these cytokines. Psoriatic lesions which cultured at the air/liquid interface in serum-free William's medium supplemented with psoriatic T cells and streptococcal antigens after 6 days still maintained the same high level in expression of the cytokines, whereas the contents of cytokines decreased obviously in the lesions which cultured by serum-free William's medium and medium added psoriatic T cells merely(P>0.05).The normal skins which cultured at the air/liquid interface in serum-free William's medium supplemented with psoriatic T cells and streptococcal antigens after 6 days have express interferon-gamma, tumor necrosis factor-alpha and interleukin-8 abnor- mally(P<0.05).Conclusion:1. The in vitro psoriatic organ culture model may serve as a valuable tool for psoriasis research. Simulated human psoriatic pathogenesis, psoriatic lesions were cultured at the air/liquid interface in serum-free William's medium supplemented with psoriatic T cells and streptococcal antigens can maintain the psoriatic structure and characterization.2. It was the first time to culture psoriatic lesions in serum-free William's medium and purified the environment for living organism culturing.3. The facility which devised for culturing the psoriatic skins supplied an air/liquid interface. This facility is a convenient and cheap tool and can be utilized for the culture of skin.4. Keratin 17 was detected that over-expressed in psoriatic skins and unexpressed in normal control groups by immunohistochemistry method and western blotting. The results confirmed the success of in vitro psoriatic organ culture model; Besides, Keratin 17 was established as psoriatic classical marker for detection.5. The expressions of interferon-gamma, tumor necrosis factor-alpha and interleukin-8 were detected by enzyme-linked immunosorbent assay within different cases and groups and analyzed all the data by SPSS and EXCEL statistically which confirmed the success of in vitro psoriatic organ culture model as well. The contents of cytokine are abnormal compared with normal human skins'. The theory that streptococcus hemolyticus stimulates the activation of T cell and interferon-gamma, tumor necrosis factor-alpha and interleukin-8 play a primary role in psoriatic pathogenesis was been confirmed.6. The essential functions of T cells and streptococcal antigens in psoriatic pathogenesis were confirmed according to the process of establish the in vitro conditions for psoriatic lesions. Observations of psoriatic characterizations and detections of some special psoriatic indexes supported the theory. Streptococcal antigens were treated as a crucial factor in psoriatic pathogenesis and T cells can been stimulated by the streptococcal antigen to form psoriatic characterization.