人视网膜色素上皮细胞吞噬过程中环磷酸腺苷及蛋白激酶C的改变

The alteration of cyclic adenosine monophosphate and protein kinase C in the phagocytic process of human pigment epithelial cells

目的 研究人视网膜色素上皮 (HRPE)细胞特异性及非特异性吞噬过程中环磷酸腺苷(cAMP)浓度及蛋白激酶C(PKC)活性的变化 ,探讨cAMP及PKC在HRPE的吞噬功能中所起的作用。方法 用 1× 10 7个 /ml视杆细胞外节膜盘 (ROS)及乳胶微球 (LB)于 37℃孵育培养的正常HRPE细胞 ,在孵育的不同时间 (5min至 4 8h)终止吞噬反应。用双重荧光标记法检测HRPE细胞吞噬动力学。用扫描及透射电镜证明HRPE细胞对LB及ROS的结合与吞噬作用。分别用12 5I cAMP放射免疫试剂盒及液闪记数γ 3 2 P放射活性法检测相应时间点的HRPE细胞特异性及非特异性吞噬时的cAMP浓度及PKC活性。结果 HRPE细胞特异性吞噬过程中 ,孵育 15min时ROS结合于HRPE细胞表面 ,此时cAMP浓度开始降低 ;胞质及胞膜PKC活性的降低早于cAMP ,发生在孵育 5min时 ,但二者均在孵育2 4h达到最低值。在非特异性吞噬过程中 ,HRPE细胞结合LB发生在孵育 90min ,在孵育的 12h内cAMP浓度无明显变化 (P >0 .0 5 ) ,12～ 4 8hcAMP浓度降低 ;胞质及胞膜PKC活性在孵育的 5min至4 8h时间内 ,与对照组比较 ,差异均无显著意义 (P >0 .0 5 )。结论 cAMP及PKC对HRPE细胞特异性吞噬吞入过程的维持十分重要 ,但不参与非特异性吞噬的直接调节。并且 ,HRPE细胞特异性吞噬过程伴随着cAMP?

Objectiv To investigate the role of cyclic adenosine monophosphate (cAMP) and protein kinase C (PKC) in the phagocytic process of human retinal pigment epithelial (HRPE) cells by measuring phagocytosis indexes in specific and nonspecific phagocytic process. Method The cultured HRPE cells were incubated with 1×10 7/ml rod outer segments (ROS) or latex beads(LB) at 37℃, then the phagocytosis were terminated at different incubation time (5 min-48 h). The kinetics of phagocytosis was measured by double- fluorescent vital assay. The binding and ingestion of ROS or LB were proved by scanning and transmission electron microscope. cAMP level was measured using 125 I-cAMP radio-immunity kit,and PKC activity was measured by counting γ- 32 P radio-activity with liquid scintillation. Result In specific phagocytosis of HRPE cells,the binding of ROS was happened at 15 min of incubation,and the cAMP level was decreased at the same time;the activity of PKC (both in cytoplasm and membrane)was decreased at 5 min which was earlier than the changes of the level of cAMP ,but both indexes were reached their lowest level at 24 h. In nonspecific phagocytosis of HRPE cells ,the binding of LB was happened at 1.5 h;on the other hand, when HRPE cells were incubated with LB, cAMP level did not change until 12 h ( P > 0.05),but it began to decrease in the incubation time from 12 h-48 h;the activity of PKC(both in cytoplasm and on membrane) was stable during all incubation periods( P >0.05). Conclusion cAMP and PKC are very important for sustaining the ingestion process in specific phagocytosis of HRPE cells, but have no direct relationship with nonspecific phagocytosis. Furthermore, cAMP level and PKC activity in HRPE cells are decreased during specific phagocytosis.