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目的 探讨核因子 κB(NF κB)在蛋白激酶C(PKC)对豚鼠哮喘模型T淋巴细胞功能调控的信号传导中的作用。方法 分别从哮喘组和正常组的豚鼠的支气管肺泡灌洗液中分离出T淋巴细胞并分别加入PKC激动剂 12 肉豆蔻酰 13 乙酸佛波酯 (PMA)和NF κB抑制剂二硫代氨基甲酸吡咯烷 (PDTC)培养。NF κB的表达、IL 4和IL 5的mRNA和蛋白质的表达、细胞增殖和凋亡分别用免疫组织化学、原位杂交、ELISA用四甲基偶氮唑盐 (MTT)微量比色法和原位末端终止法 (TUNEL)检测。结果 加入PMA培养的哮喘组T淋巴细胞NF κB的活化、细胞增殖反应、IL 4和IL 5的mRNA和蛋白质的表达均显著高于其空白对照 (P <0 0 1) ,而同时加入PMA和PDTC培养的哮喘组T淋巴细胞以上指标均显著低于只加入PMA培养的哮喘组T淋巴细胞 (P <0 0 1) ;加入PMA培养的哮喘组T淋巴细胞的凋亡指数显著低于其空白对照 (P <0 0 1) ,而同时加入PMA和PDTC培养的哮喘组T淋巴细胞凋亡指数显著高于只加入PMA培养的哮喘组T淋巴细胞 (P <0 0 1)。T淋巴细胞NF κB的活化与增殖反应呈显著正相关 (r =0 6 4,P <0 0 0 1) ,与IL 4和IL 5的mRNA和蛋白质的表达也均呈显著正相关 (r=0 5 5 - 0 6 8,P <0 0 0 1) ,而与凋亡指数呈显著负相关 (r =- 0 6 2 ,P <0 0 0 1)。结
Objective To investigate the role of nuclear factor κB (NFκB) in the signal transduction of protein kinase C (PKC) on the functional regulation of T lymphocytes in guinea pig asthma model. Methods T lymphocytes were isolated from bronchoalveolar lavage fluid of guinea pigs in asthmatic group and normal control group, and were respectively treated with PKC agonist 12 phorbol myristate acetate (PMA) and NF κB inhibitor dithiocarbamic acid Pyrrolidine (PDTC) culture. NF κB expression, IL 4 and IL 5 mRNA and protein expression, cell proliferation and apoptosis were detected by immunohistochemistry, in situ hybridization, ELISA with MTT micro-colorimetry and the original TUNEL assay. Results The activation of NF-κB, the cell proliferation, the mRNA and protein expressions of IL-4 and IL-5 in PMA-cultured asthma group were significantly higher than those in the blank control group (P <0.01) The above indexes of T lymphocytes in asthmatic mice cultured with PDTC were significantly lower than those in asthmatic mice with PMA alone (P <0.01). The apoptotic index of T lymphocytes in asthmatic mice treated with PMA was significantly lower than that of blank (P <0.01). However, the apoptosis index of T lymphocytes in asthmatic mice treated with PMA and PDTC was significantly higher than that of T lymphocytes cultured in PMA alone (P <0.01). There was a significant positive correlation between the activation of NF-κB and the proliferative response of T lymphocytes (r = 0 64, P 0 01), as well as the mRNA and protein expressions of IL 4 and IL 5 (r = 0 5 5 - 0 6 8, P 0 01 0), but negatively correlated with the apoptotic index (r = - 0 6 2, P 0 01 0). Knot