A Study On Immunomodulation Of Autocrine Prolactin By RNAi-mediated Silencing Of Prolactin Receptor In Activated Jurkat Cells

Objects: A great number of literatures have suggested that endocrine plays an important role in the immune response. However, the immunomodulation of autocrine prolactin in T lymphocytes remains unclear. In order to study the action of autocrine prolactin in activated T lymphocytes, we constructed a cell model with PRLR knockout lentivirus-mediated RNA interferent and assay the change of its immunoreation to PHA. In this study, we first designed and constructed the cell model with PRLR knockout successfully. Secondly, we investigated the influence of autocrine prolactin about the proliferation of Jurkat cells with PRLR knockout and the molecular mechanism of it. The third, we evaluated how the autocrine PRL regulates homeostasis between Th1 and Th2 activity and the expression of IL-10.Methods: 1. A short hairpin RNA construction as homologous PRLR RNA succession was designed to cloned into lentivirus vector using Elbashi's criteria and RNAi vector rule; then the recombinant was evaluated by enzyme cutting and gene sequencing test; at last it was packaged into lentivirous and its tite was assayed; 2. The Jurkat cells were infected by lentivirus. The infection was observed under fluorescence microscropy. The effect knockout was analyzed by western blot; 3. The Nb2 lymphocyte bioassay was used to assay the level of autocrine PRL; 4. MTT assay and cell count were used to evaluate the proliferation of Jurkat cells without PRLR, which were stimulated by PHA. And its mechanism was studied by assaying the expression of anti-apoptosis gene BCL-2; 5. The expressions of CD28,CD154 and CD137 on Jurkat cells with PRLR knockout were detected with flow cytometry; 6.The levels of IL-2,IL-4,IFN-γand IL-10 were measured with two steps sandwich ELISA.Results: 1. We successfully constructed a recombinant specifically targeting PRLR.The recombinant was checked with enzyme cutting and gene sequencing test; 2. After the Jurkat cells were infected for 168h, the expression of PRLR was checked to be suppressed significantly with western blot; 3. The bioassay was specificity and sensitivity, and knockout of PRLR up-regulated the secretion of autocrine PRL; 4. The proliferation of Jurkat cells with PRLR knockout was down-regulated because of decreasing expression of the protein Bcl-2; 5. Costimulatory molecules CD154 and CD137 were down-regulated compared to the control,but CD28 was not influenced. 6. The secretion of IL-2 and IL-4 was decreased (P<0.05), but IFN-γand IL-10 were not changed.Conclusions: 1. The Jurkat cell with PRLR knockout by lentiviral shRNA interference can keep stable silencing of PRLR to hamper the action of autocrine prolactin. The constructed cell model is useful to study the action of autocrine prolactin on T lymphocytes; 2. The secretion of autocrine prolactin increased in the Jurkat cells with PRLR knockout. It suggestes that autocrine PRL is negtively regulated by PRLR; 3. The autocrine prolactin acts as a cofactor in the proliferation of T lymphocytes. The mechanism is that it can modulate the expression of Bcl-2; 4. The autocrine PRL also affected the quality of immune response through influencing the expression of costimulatory molecules and cytokine secretion; 5. The autocrine PRL modulates the balance of Th1 and Th2 cytokines, not the immunomodulatory molecules IL-10; 6. The autocrine prolactin in the microenviroment of T lymphocytes plays an important role in the survival of T lymphocytes and the lymphocyte response.