单克隆抗体HIM82对人中性粒细胞钙离子和蛋白激酶及酪氨酸激酶信息途径的降调节作用

Down regulation effect of McAb HIM 82 on Ca 2+ , protein kinasec, protein tyrosine kinase signal pathway of neutrophils

目的 研究单克隆抗体（ 单抗） Ｈ Ｉ Ｍ８２ 对中性粒细胞（ Ｎｐ） 呼吸爆发产生的活性氧物质（ Ｒ Ｏ Ｓ） 的调控作用，并探索 Ｈ Ｉ Ｍ８２ 对该调控作用相关联的信息传递途径的影响。方法 比较对照组与实验组（ 单抗结合组） 在趋化物激活后， Ｒ Ｏ Ｓ水平、对 Ｇ 蛋白抑制的影响、胞内［ Ｃａ２ ＋ ］ 水平、蛋白激酶 Ｃ（ Ｐ Ｋ Ｃ） 、酪氨酸激酶（ Ｐ Ｔ Ｋ） 活性的差异而判别该单抗对 Ｒ Ｏ Ｓ 及有关信息途径的影响。结果 Ｈ Ｉ Ｍ８２ 与 Ｎｐ 结合的阳性率达９５ ％ 以上（ 对照组为０） ，以３０μｇ／ｍｌ Ｈ Ｉ Ｍ８２ 作用 Ｎｐ 再以佛波酯激活则对产生的 Ｏ２ － 、 Ｈ２ Ｏ２ 分别下调５５ ％ （ Ｐ＜ ００１） 和３２ ％ （ Ｐ＜ ００１） ，对趋化三肽（ Ｆ Ｍ Ｌ Ｐ） 、 Ｐ 物质激活 Ｎｐ 产生的 Ｈ２ Ｏ２ 分别下调１６ ％ （ Ｐ＜ ００１） 和３７ ％ （ Ｐ＜ ００１） 。 Ｈ Ｉ Ｍ８２ 的同系单抗 Ｈ Ｉ Ｍ７０ 对上述活化 Ｎｐ 产生的 Ｏ２ － 、 Ｈ２ Ｏ２ 也有相似的降调节作用。用参比法观察 Ｈ Ｉ Ｍ８２ 对 Ｇ 蛋白活性影响的实验中，发现 Ｇ 蛋白抑制剂百日咳毒素（ Ｐ Ｔ） 对 Ｆ Ｍ Ｌ Ｐ激发生成的 Ｈ２ Ｏ２ 抑制率为４２ ％ ， Ｈ Ｉ Ｍ８?

Objectives To study the control effects of HIM 82 and HIM 70 on reactive oxygen species (ROS) released from respiratory burst of neutrophils (Np), and the action of HIM 82 on relevant signal transduction pathway Methods The experimental group (combining with McAb) was compared with the control group with regard to the ROS level, activity of G protein, intracellular [Ca 2+ ] level, the activities of PKC and PTK in activated Np to interpret the mechanism by which the McAb down regulated the respiratory burst Results The positive rate of Np combined with HIM 82 was shown over 95% using indirect immunofluorescence assay (control group was 0) The generation of superoxide anion and hydrogen peroxide from PMA treated Np were decreased by 55%( P <0 01) and 32%( P <0 01)with 30 μg/ml HIM 82 The amount of H 2O 2 produced in FMLP or SP stimulated Np were decreased by 16%( P <0 01) and 37%( P <0 01) respectively A similar effect of HIM 70 on Np was observed Further more, the inhibition of H 2O 2 in FMLP activated Np affected by PT, a specific inhibitor for G protein, and in HIM 82 was 42% and 60% separately, and that with both PT and HIM 82 was 85% It was suggested that HIM 82 could enhance the inhibition effect of PT on G protein The intracellular [Ca 2+ ] level of Np stimulated with FMLP was decreased by 85%, and that of Np activated with PMA by 100% nearly in the present of HIM 82 The activities of PKC and PTK in PMA stimulated Np were decreased by 23%( P <0 05) and 35%( P <0 05) separately when treated with HIM 82 Conclusion The McAb HIM 82 could down regulate the ROS produced from activated Np. The mechanism of decreasing ROS may be involved in down regulating Ca 2+ PKC and PTK signal transduction pathway activities that are upstream control systems for NADPH oxidase activation