hsbaff上调胞内钙离子调控cd4t淋巴细胞增殖和活性机理研究-study on the mechanism of hs baff up-regulating intracellular calcium to regulate cd4t lymphocyte proliferation and activity.docx

Summarywith hsBAFF (0.5, 2.5 μg/ml) with/without anti-CD3 (1 μg/ml), or in the presence or absence of IL-2（50 and 100 U/ml）or IFN-γ（1 ng/ml）for 12, 24, 72 h. CD4+ T cellproliferation was evaluated using an MTT assay. IL-2 and IFN-γ levels was measured by 125I based radioimmunoassay (RIA) and a sandwich ELISA reagent, respectively. The Results showed that anti-CD3 only or anti-CD3+hsBAFF significantly elevated CD4+ T cell proliferation. hsBAFF significantly increased IL-2 and IFN-γ levels in the culture supernatants of CD4+ T lymphocytes. Coordination of IL-2 or INF-γ with hsBAFF regulated the proliferation and high INF-γ or IL-2 level in CD4+ T lymphocytes. Our data clearly indicate that hsBAFF may enhance the proliferation and cytokine IL-2 and IFN-γ secretion in CD4+T lymphocytes, and IL-2 or INF-γ may coordinate hsBAFF to better regulate function activity in CD4+ T lymphocytes.3Discussion on hsBAFF-upregulated [Ca2+]i homeostasis regulates function activity in CD4+T lymphocytesTo study the effect of hsBAFF on intracellular free Ca2+ concentration ([Ca2+]i) of in vitro mouse splenic CD4+ T lymphocytes and to discuss the cytological mechanism of hsBAFF-regulated CD4+ T lymphocyte proliferation. 1) The isolated CD4+ T cells were purified by using immunomagnetic beads. Cell suspensions (3×106/ml) were seed in 24-well flat-bottomed plates and treated with hsBAFF (0, 2.5, 5, 10 μg/ml) with/without anti-CD3 (1 μg/ml) for 12 h, and then loaded with fluorescence probe Fluo-3/AM. Fluorescence intensity of [Ca2+]i in CD4+ T lymphocyte were assayed via flow cytometry. 2) Cell suspensions were seed in 96-well flat-bottomed plates and treated with hsBAFF (0, 0.5, 2.5 μg/ml) with/without anti-CD3 (1 μg/ml) for 72 h following pretreatment with 20 μM BAPTA/AM for 1 h, or were treated hsBAFF (0, 0.5, 2.5 μg/ml) with/without anti-CD3 and with/without 0.5 μM Tg for 24h or 48 h. Cellular proliferation and viability were evaluated using an MTT assay. The Results showed that Fluo