The Experiment Study Of Co-cultured T Lymphocytes And BV-2 Microglia Cells After Thymus Immune Tolerance

Objective: The interaction between activated microglia and T lymphocytes can yield abundant pro-inflammatory cytokines. Our previous study proved that thymus immune tolerance could alleviate the inflammatory response. This study aimed to investigate whether intrathymic injection of myelin basic protein(MBP) in mice could suppress the inflammatory response after co-culture of T lymphocytes and BV-2 microglia cells.Methods: Totally, 72 male C57BL/6 mice were randomly assigned to three groups(n = 24 in each): Group A: intrathymic injection of 100 ?l MBP(1 mg/ml); Group B: intrathymic injection of 100 ?l phosphate-buffered saline; and Group C: sham operation group. Every eight mice in each group were sacrificed to obtain the spleen at postoperative days 3, 7, and 14, respectively. T lymphocytes those were extracted and purified from the spleens were then co-cultured with activated BV-2 microglia cells at a proportion of 1:2 in the medium containing MBP for 3 days. After identified the T lymphocytes by CD3, surface antigens of T lymphocytes(CD4, CD8, CD152, and CD154) and BV-2 microglia cells(CD45 and CD54) were detected by flow cytometry. The expressions of pro-inflammatory factors of BV-2 microglia cells(interleukin [IL]-1?, tumor necrosis factor-? [TNF-?], and inducible nitric oxide synthase [i NOS]) were detected by quantitative real-time polymerase chain reaction. One-way analysis of variance and the least significant difference test were used for data analysis.Results:(1) Compared with Group B and C, the ratio of CD4+/CD8+ T in Group A showed a downward trend from the 3rd to 7th day, with the minimum at postoperative day 7, then an upward trend from the 7th to 14 th day(P < 0.05).(2) The levels of CD152 in Group A showed an upward trend from the 3rd to 7th day,with a downward trend from the 7th to 14 th day(20.12 ± 0.71%, 30.71 ± 1.14%, 13.50 ± 0.71% at postoperative days 3, 7, and 14, respectively, P < 0.05). Meanwhile, the levels of CD154 in Group A showed a downward trend from the 3rd to 7th day, with an upward trend from the 7th to 14 th day(10.00 ± 0.23%, 5.28 ± 0.69%, 14.67 ± 2.71% at postoperative days 3, 7, and 14, respectively, P < 0.05).(3) The levels of CD45 and CD54 in Group A were found as the same trend as the ratio of CD4+/CD8+ T(CD45: 83.39 ± 2.56%, 82.74 ± 2.09%, 87.56 ± 2.11%; CD54: 3.80 ± 0.24%, 0.94 ± 0.40%, 3.41 ± 0.33% at postoperative days 3, 7, and 14, respectively,P<0.05).(4)The expressions of TNF-?, IL-1?, and i NOS in Group A were significantly lower than those in Groups B and C, and the values at postoperative day 7 were the lowest compared with those at postoperative days 3 and 14(P < 0.05). No significant difference was found between Groups B and C.Conclusions: Intrathymic injection of MBP could suppress the immune reaction that may reduce the secondary immune injury of brain tissue induced by an inflammatory response.