The Immunity Modulatory Effects Of Prolactin And Proteomics Analysis The Signal Transduction Pathways Of Prolactin

Objects: The experiment was designed to investigate three aspects of the immunity modulatory effects of prolactin, which included the expressions of co-stimulatory molecular CD154 and CD28 on JurkatD1.1 cells, the proliferation of JurkatD1.1 cells , and the shift of the balance of Th1/Th2 cytokines. Furthermore, the feasibility of appling proteomics to analysis the profile of the network of the signal transduction pathways of prolactin was investigated.Methods: In the first part of the experiment, JurkatD1.1 cells were cultured in the presence of recombinant human prolactin(rhPRL) or phytahematoagglutinin(PHA) alone and combination of them. The expressions of CD154 and CD28 moleculars on JurkatD1.1 cells were detected with flow cytofluorometer. Dose-dependent study and time-dependent study were also carried out. Prolactin receptor monoclonal antibody(PRLrAb) was then added into the medium to block the effects of prolactin, and the expressions of CD154 and CD28 moleculars were compared among the above groups. Meanwhile,semi-quantitative RT-PCR was applied to determine the levels of mRNA of CD154 and CD28 moleculars in each group. In the second part, cell count and MTT assay were used to elevate the proliferation of JurkatD1.1 cells stimulated by rhPRL for 72 hours. In the third part, JurkatD1.1 cells were stimulated with different concentrations of rhPRL for 24 hours. Then the levels of IFN-γand IL-4 in the supernatants were measured with enzyme linked immunosorbent assay(ELISA), and the ratio of IFN-γ/IL-4 was calculated. In the last part, phosphate metal affinity chromography(PMAC) was applied to enrich phosphoproteins, and which were resolved by one-dimensional SDS-PAGE (1DE). Immage scanner was used to dectect different bands between control group and rhPRL stimulation group, and which were...