| Psoriasis is a common chronic inflammatory skin disease, which has genetic predisposition..Foreign antigens stimulate the clusters of differentiation 4+ (CD4+) T cells and stimulated cells could secrete Interleukin (IL)-2,6,8.At the same time, these factors could stimulate keratinocyte (KC) proliferation. Stimulated KC could secrete IL-1,6,8 and these factors could effect on CD4+T cells again or IL-6,8 could effect on KC themselves. In addition, tumor necros factor- alpha (TNF-α) and gamma Interferon (IFN-γ) secreted by the activated CD4+T cells make KC to secrete intercellular adhesion molecule (ICAM). The ICAM make KC to be adhered to CD4+T cells, which cause pathological changes happen again and again.Hedgehog (Hh) signal pathway is a signal transduction pathway that can regulate embryonic development. After the completion of embryonic development, it could continue to regulate cell growth, proliferation and differentiation. Abnormal activation of Hh signal pathway can make KC express some pathology features of psoriasis, such as the KC proliferation, differentiation abnormal keratin (K) 14 expression levels increased, epidermal growth factor receptor (EGFR) phosphorylation increase and so on. Cyclopamine that the signal pathway efficient and specific inhibitor can be quickly eliminate d psoriatic lesions and shows a significant effect in psoriasis therapy.We have found that Hh signaling pathway is in a state of excessive activation in the psoriatic lesions.To further study the mechanism of cyclopamine to treat the psoriasis, we use a SCID-hu xenogeneic transplantation model of psoriasis and the normal keratinocytes (Hacat) in this experiment and observe skin graft pathological changes and Hacat secrete and express after cyclopamine cytokine effect on them. Through this method, we can understand the mechanism of Hh pathway in psoriasis and approach the mechanism of cyclopamine therapy in psoriasis.The main experimental methods and results are described as follows:Part 1 The production of the SCID-hu xenogeneic transplantation model Psoriatic skin that obtained from psoriasis lesions was transplanted onto the back of the SCID mice. After 10 days later, concanavalin (ConA)-stimulated peripheral blood monouclear cells (PBMCs) (approximately 4×106 cells for each mouse) were injected subcutaneously under the grafted skin. After 15 days transplantation, the majority of skin graft has significantly appearance and histopathologic features of psoriasis. We use Baker score system to score all transplant models. The result is 8.278±0.244 (the typical lesion score in psoriatic patients is between 4.0-10.0). It is therefore indicated that the SCID-human skin chimeric model with clinical, pathology characteristics of psoriasis was successfully constructed.Part 2 The effects of cyclopamine on psoriasis in animal model 1 Division and administrationExperimental mice were divided into 3 groups, each group with four mice. In the 5mmol/mL cyclopamine treatment group, cyclopamine, diluted by DMSO and normal saline, was delivered transcutaneously at a dose of 50μL for each administration, applied twice every 1 days, for 15 days of treatment; 5mmol/mL tomatidine control group; tomatidine replace cyclopamine, dosage, method of preparation and external application the same as cyclopamine; blank control group: DMSO, normal saline, usage same as above.2 ResultsEach group therapeutic efficacy was compared after treatment for 15 days:The psoriatic lesions were improved gradually by cyclopamine treatment. Skin graft appearance showed that the skin graft erythema, Linxie disappeared and almost closed to normal skin; Histological appearance of grafts showed the thining of the epidermis, reducing of lymphocyte infiltration in the dermis and decreasing of histological score. In contrast, no significant improvement was noticed in tomatidine control group and blank control group. Histological appearance of grafts showed the epidermis was still thick and the features of psoriasis including hyperkeratosis, acanthosis with elongation of rete pegs and lymphocytic infiltration were still observed in control groups, although the appearance of the granular layer concomitant with the disappearance of parakeratotic changes was notable in some of the lesions.In conclusion, by using cyclopamine treatment on psoriasis in SCID-hu mouce model, we found that it could significantly promote SCID mice psoriatic lesions recovery and it has excellent therapeutic efficacy for psoriasis. Furthermore, cyclopamine is hopeful to be developed as new clinical therapies for psoriasis in the near future. This study lays the foundation on clinical application of cyclopamine and supplys a new direction for the treatment of psoriasis.Part 3 The cytokine secrete change of Hacat after using the cyclopamine. By using ELISA and RT-PCR method, we detect the expression of IL-6, 8, TNF-αof Hacat after cyclopamine effect. Results are as follows: 1 By using ELISA to detect cell culture supernatant of IL-6, 8, TNF-αsecretion after Hacat handles with cyclopamine at 12h, 24h, 48h, we find the experiment group is significantly lower than the control group. They have statistics significance, especially in 5μmol/mL cyclopamine group.2 The group of Hacat handled with cyclopamine at 12h, 24h, 48h after using RT-PCR method to detect cell IL-8 mRNA expression levels were significantly decreased, especially in 5μmol / mL cyclopamine group.Cyclopamine could significantly reduce the secretion and expression of IL-6,8,TNF-α, which suggest cyclopamine has the good effect on treatment of psoriasis. One of likely reason is the cyclopamine could reduce the secretion of IL-6, 8, TNF-αthat play an important role in the pathogenesis of psoriasis. |
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