摘要
利用免疫印迹技术及内源性底物磷酸化方法,我们研究了在巨噬细胞的信号传递中起重要作用的PKC同功酶的分布及其在免疫调变剂LPS的刺激下产生的激活和转位。在未激活的巨噬细胞中,PKC-β的含量高于PKC-α和ε,它和PKC-α的分布均是胞质中大于胞膜。以PMA为阳性对照,结果提示LPS介导的抑制性巨噬细胞兔疫调变机制中涉及到了PKC-α和PKC-ε从胞质到膜组份的转让而不是PKC-β(PKC-βⅠ或βⅡ)的转位。应用PKC-依赖的内源性底物磷酸化的分析,结果说明胞质中55 kDa和74 kDa蛋白质磷酸化条带的减弱和膜组份中的类似蛋白质(PMA与LPS的刺激有不同的反应)磷酸化强度的增加,与PKC的转位有着相同的时间梯度。这结果提示在LPS介导的免疫调变信号传递通路中,PKC-α和PKC-ε可能介导了信号的传导及放大。
Suppressor macrophages induced by the continuous invasion of tumor cells and parasites, which can acquire an ability in vitro to kill or inhibit tumor cells and inhibit the activity of T, B lymphocytes and NK cells, have been indicated. We have developed a procedure previously to modulate the suppressor macrophages by bacterial lipopolysaccharide (LPS). The modulated macrophages remained and even enhanced the ability to inhibit tumor growth and to up-regulate or enhance the activities of T, B lymphocytes and NK cells in vitro. However, the mechanisms of macrophage modulation by LPS are unknown. This investigation was designed to analyze the regulation of PKC activity and to characterize the isoforms of PKC during macrophage modulation by using Western blot and endogenous substrate phosphorylation (PKC-DESP). In
rest cells, PKC-β was found to be the most abuncant isoform in macrophages: and PKC-α,β was found predominantly in the cytosol. Using PMA as a positive control, we found that the immuno-mo-dulator agent- LPS triggered the physical translocation from the cytosol onto the membrane of PKC-α and PKC-ε, but PKC-β(βⅠ or βⅡ ) was difficult to detect. The analysis of PKC-DESP showed a pattern with a time course similar to that observed with Western blot. We observed that LPS and PMA increase the level of phosphorylation of 55 kDa and 74 kDa proteins with a corresponding decrease in the cytosolic proteins. It suggests that the translocation of PKC-α and PKC-ε, may be important events involving in the PKC-pathway by LPS-mediated modulation in suppressor macrophages.
出处
《实验生物学报》
CSCD
1996年第4期429-434,共6页
Acta Biologiae Experimentalis Sinica
基金
中国科学院上海生命科学联合开放实验室
自然科学基金
关键词
细胞
巨噬细胞
蛋白激酶C
脂多糖
佛波脂
Inlmunornodulation of suppressor macrophages. Signal transduction. Activation and translocation of PKC.
