Study On The Role Of RasGRP1in The Pathogenesis Of Eczema And Mechanisms

Part I The expressions of RasGRPl in peripheral blood lymphocytes and T cells of eczema patientsObjective To study the expressions of RasGRP1in peripheral blood mononuclear cells (PBMCs) of patients with eczema. To purify T cells from PBMC and evaluate the expressions of RasGRP1in T cells of eczema patients. Furthermore, to explore its significance.Methods Human PBMCs in eczema patients and the normal subjects were extracted by Ficoll extractionl. T cells were separated from PBMCs by means of immunomagnetic beads. The purity of separation was validated and the expressions of RasGRP1in T cells of eczema patients and the normal subjects were detected by RT-PCR and Western Blot.Results The purity of PBMC was close to100%by Ficoll extraction, while the purity of T cell could reach more than98%by immunomagnetic separation. In PBMCs and the purified T cells, the expressions of RasGRP1in eczema patients were higher than those in the normal subjects.Conclusions T cells with high purity could be acquired by the use of immunomagnetic separation. The expressions of RasGRP1in both PBMC and T cells of eczema patients were higher than the normal subjects. Aberrant expressions of RasGRP1might contribute to the pathogenesis of eczema. Part II The effects of dexamethasone on the expressions of RasGRPl in T cells of eczema patientsObjective To study the effects of dexamethasone on the expression of RasGRP1in T cells of eczema patients. To select the optimum concentration of dexamethasone and investigate its effects on T cell function.Methods Patients with severe eczema using dexamethasone were selected. The expressions of RasGRP1in T cells before and after dexamethasone therapy were detected. Dexamethasone was dubbed in three different concentrations of1×10-5mol/L,1×10-6mol/L and1×10-7mol/L. Dexamethasone co-cultured with T cells in eczema patients without dexamethasone therapy. The expressions of RasGRP1were detected by RT-PCR. To observe the expressions of RasGRP1in T cells at different time points (Oh,6h,12h,18h,24h) of dexamethasone. After the stimulation of different concentrations of dexamethasone, the apoptosis of T cells were detected by MTT.Results Without dexamethasone therapy, The expressions of RasGRP1in patients with eczema were higher than those in normal subjects. One week later by the use of dexamethasone therapy, the expressions of RasGRP1decreased significantly, which were close to the normal expression levels. Dexamethasone could inhibit the expressions of RasGRP1in eczema patients with three different concentrations of dexamethasone.1×10-5mol/L concentration had the best inhibitory effect on the expressions of RasGRPl. When using the concentration of1×10-5mol/L to inhibit T cell, the expressions of RasGRP1decreased with time prolonging. The inhibitory effect of dexamethasone was strongest after24h. Different concentrations of dexamethasone could promote apoptosis of T cells. And the apoptosis in a concentration of1×10-5mol/L dexamethasone was the strongest.Conclusions Dexamethasone could inhibit the expression of RasGRP1in peripheral blood T cells of the patients with eczema and promote the apoptosis of T cells. The effects of inhibition increased with the increase in the concentration of dexamethasone and time. One week After the use of dexamethasone, the level of RasGRP1in peripheral blood T cells of eczema patients decreased to the normal levels. This may be a new mechanism for anti-inflammatory effects of dexamethasone. Part Ⅲ Construction of lentiviral vectors containing H-RasGRPl-shRNA and its effects on T cellsObjective To construct lentiviral vectors containing the gene of H-RasGRP1-shRNA and transfect T cells. To explore the expressions of RasGRP1in T cells after RNA interference. To study the effects on Ras downstream signaling proteins, apoptosis and cytokine secretion after silencing RasGRP1gene. And to investigate the mechanisms in T cells.Methods Lentiviral vectors containing the gene of H-RasGRP1-shRN A were constructed and the viral titer1.0×108TU/ml was acquired. When T cells were transfected by lentiviral vectors, the expressions of RasGRP1in T cells were detected by RT-PCR and Western Blot methods at different time points (12h,24h,48h,72h). Apoptosis of T cells were detected by flow cytometry. After PMA and ionomycin stimulated T cells, the expression of Ras protein downstream signaling pathways-p-Erk, Erk, Akt and p-Akt were observed. To detect T cell cytokine secretion of IL-2, IL-4and IL-17by the method of ELISA. Lentiviral vectors were transfected into PMA and ionomycin stimulated T cells. The expressions of p-Erk, Erk, Akt and p-Akt protein in transfected T cells, as well as T cells cytokines were detected.Results H-RasGRP1-shRNA lentiviral vectors were constructed successfully and its biological titer reached1.0×108TU/ml, which could be used for gene delivery. After transfection of T cells by lentiviral vectors, the expression of RasGRP1decreased in the mRNA and protein levels. With the extension of time (12h,24h,48h,72h), RasGRP1expression levels gradually decreased and expression of RasGRP1almost disappeared at72h. Apoptosis of T cells accelerated obviously. The expressions of p-Erk, Erk, Akt and p-Akt were found in T cells of both eczema patients and the normal subjects. And the expressions in eczema patients were higher than those in the normal subjects. After the stimulation PMA and ionomycin on T cells, the expressions of p-Erk, Erk, Akt and p-Akt were significantly increased, especially in eczema patients. In addition, the levels of T cell cytokine IL-2, IL-4and IL-17were significantly increased. After lentiviral vectors transfected PMA and ionomycin-stimulated T cells, the expressions of p-Erk and p-Akt decreased, while the expression of Erk and Akt did not change significantly. The levels of cytokines IL-2, IL-4and IL-1in T cell culture supernatants were on the decrease.Conclusions Lentiviral vectors containing H-RasGRP1-shRNA had been constructed successfully, which could meet the requirement of transfection. Lentiviral vectors could be successfully transfected to T cells and inhibit the expressions of RasGRP1and apoptosis in T cells. When the eczema occurs, T cell receptor activation may be mediated by the activation of RasGRP1expression. The downstream signaling molecule of Ras protein-Erk and Akt phosphorylates, leading to the increasing of T cell cytokine. Therefore, RasGRP1may be a possible target in eczema treatment. It provides a novel idea for gene therapy and immune therapy.