| Objective: Observe the effects of four kinds of immunosuppressants(dexamethasone,cyclophosphamide, Cyclosporin A and mycophenolate mofetil) on the expression of Dectin-1/TLR2 receptor and the release of TNF alpha in RAW264.7 cell line induced by Zymosan A. By fungal infections in vitro test, from the pattern recognition mechanism to explore the influence of different immune inhibitors on natural immune system to recognize fungal pathogens, and further reveal the relationship between different immunesuppressors and fungal infection, as well as the difference among them.Methods: 1. In vitro culturing RAW264.7 macrophages, 100 μg/ml Zymosan A intervention for cell stimulation, RT-PCR technique and flow cytometry were used respectively to detect TLR2/Dectin-1 m RNA and protein level of change. ELISA was used to detect the change of inflammatory factor TNF alpha. 2. The cells were given different doses of dexamethasone and cyclophosphamide, Cyclosporin A and mycophenolate mofetil for 24 h. To detect the influence of different immunosuppresants on cell proliferation by CCK-8 assay, and choose the appropriate drug concentration for subsequent tests. 3. Dectin-1, TLR2 m RNA and protein levels of change were detected with RT-PCR technique and flow cytometry 24 hours after the intervention of dexamethasone, cyclophosphamide, Cyclosporin A and mycophenolate mofetil. 4. After dexamethasone, cyclophosphamide, Cyclosporin A and mycophenolate mofetil intervention 24 h respectively, the cells were treated with100 μg/ml Zymosan A separately. RT- PCT and flow cytometry were used to detect the Dectin-1, TLR2 m RNA and protein levels. The levels of IL-2 and INF-y in lymphocyte cμlture supernatant were detected by ELISA. Concentration and tendency of change of TNF alphain the supernatant were tested by enzyme linked immunosorbent assay(ELISA).Resμlts: After 2 hours of Zymosan A function, the expression level of Dectin-1m RNA quickly reached the highest leveland then began to decline, but TLR2 m RNA level began to rise after 2 hours, and reached to peak after 4 hours then began to decline; After 3 hours of Zymosan A intervention, the Dectin-1 protein can be detected on the surface of RAW264.7 cells, and the content increased after 6 hours.The concentration of TNF alpha began rising to rise when 2 hours, and achieved maximum at 12 hr, then gradually reduced; 2. CCK-8 cell proliferation toxicity experiment results show that with the increase of the dose of dexamethasone,cyclophosphamide, Cyclosporin A and mycophenolate mofetil, the different degree of inhibition of RAW264.7 cell proliferation in mice can be saw after the intervention of 24 hours. 3. After 24 hours of Dexamethasone stimulation, Dectin-1 m RNA and protein level decreased, TLR2 m RNA and protein levels elevated; However Cyclophosphamide has no obvious influence on Dectin-1 and TLR2. Mycophenolate mofetil and Cyclosporin A can both inhibit the expression of Dectin-1 A and TLR2.4.For dexamethasone pretreatment macrophages stimulated by Zymosan A, the Dectin-1 and TLR2 m RNA and protein levels decreased, while the concentration of TNF alpha has no significant change compared with cell stimulated by Zymosan A alone. Cyclophosphamide had no significant effect on the expression of Dectin-1 and TLR2, but can slightly improve the TNF alpha release. Mycophenolate mofetil and Cyclosporin A can inhibit the expression of Dectin-1, TLR2 m RNA and protein induced by Zymosan A in different degrees and reduce the secretion of cytokines TNF alpha.Conclusion: Dexamethasone can inhibite the transcription and expression of Dectin-1and TLR2 induced by zymosan A; Mycophenolate mofetil and cyclosporin A both inhibite the m RNA and protein expression of zymosan A-induced Dectin-1 and TLR2;While cyclophosphamide has no effects on zymosan A-activated Dectin-1/TLR2 signaling pathway, thus the mechanism of refractory infection caused by cyclophosphamide maybe have nothing to do with it. |
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