雷帕霉素对脂多糖诱导小鼠急性肺损伤后脾γδT淋巴细胞与肺巨噬细胞相互作用的影响

目的 探讨雷帕霉素(RAPA)对脂多糖(LPS)诱导的小鼠急性肺损伤后脾γδT淋巴细胞与肺巨噬细胞相互作用的影响.方法 6～8周龄健康雄性C57BL/6小鼠24只,随机分为磷酸盐缓冲液(PBS)组、LPS组、RAPA组及LPS+RAPA组.气管内注射LPS构建小鼠急性肺损伤模型,采用免疫磁珠法提纯给药1 d后处死的小鼠脾γδT淋巴细胞和肺巨噬细胞,调整细胞浓度为106 个/ml;24孔板分成PBS、LPS、RAPA及LPS+RAPA共4组,每组又分为以下3个复孔:单独培养的脾γδT淋巴细胞、单独培养的肺巨噬细胞及按1:1混合培养的脾γδT淋巴细胞和肺巨噬细胞.24 h后采用酶联免疫吸附(ELISA)法测定培养上清液中干扰素γ(IFN-γ),肿瘤坏死因子α(TNF-α)的浓度;采用实时定量聚合酶链反应法测定培养的细胞中IFN-γ及TNF-α mRNA的表达.结果 LPS组支气管肺泡灌洗液的总细胞和淋巴细胞浓度明显大于PBS组和LPS+RAPA组(P＜0.05).脾脏γδT淋巴细胞与肺组织巨噬细胞按1:1昆合培养时,LPS组上清液IFN-γ高于其他3组(P＜0.05),而TNF-α只高于RAPA组和LPS+RAPA组(P＜0.05).混合培养时LPS组IFN-γmRNA的表达明显高于PBS组和RAPA组(P＜0.05).结论 RAPA能显著抑制小鼠LPS诱导急性肺损伤后脾脏γδT淋巴细胞和肺组织巨噬细胞混合培养时IFN-γ和TNF-α的分泌.

Abstract：

Objective To explore the influences of rapamycin (RAPA) upon the cytokine changes of activated spleen γδT lymphocytes and lung tissue macrophages in acute lung injury of mice induced by lipopolysaccharide (LPS).Methods A total of 24 healthy male C57BL/6 mice,6 -8 weeks old,were randomly divided into phosphate buffered saline (PBS),LPS,RAPA and LPS + RAPA groups.Acute lung injury was induced by a single intratracheal instillation of LPS in mice.And spleen γδT lymphocytes and lung macrophages were purified by immunomagnetic beads at Day 1.The purified spleen γδT lymphocytes and lung macrophages were adjusted to 106 cell/ml.And 24-well plates were used for 4 groups.Each group were further separated with spleen γδT lymphocytes alone,lung tissue macrophages alone and co-culturing.Supernatant fluid was collected after 24 hours.The expressions of IFN-γ and TNF-o were analyzed by ELISA (enzyme-linked immunosorbent assay).And the expressions of mRNA were analyzed by real-time quantitative PCR (polymerase chain reaction).Results The total cells numbers and lymphocytes numbers of bronchoalveolar lavage fluid were significantly higher in LPS group than those in PBS and LPS + RAPA groups (P＜0.05).And the level of IFN-γwas significantly higher in LPS group than that in PBS,RAPA and LPS + RAPA groups by co-culture (P ＜ 0.05).The level of TNF-α was significantly higher in LPS group than that in RAPA gaud LPS + RAPA groups by co-culture (P ＜ 0.05).However,the mRNA of IFN-γ was higher in LPS group than that in PBS and RAPA groups (P ＜ 0.05).Conclusion RAPA inhibits the secretion levels of IFN-γ and TNF-α in spleen γδT lymphocytes and lung tissue macrophages in acute lung injury of mice induced by LPS.