氯离子转运通道在人视网膜色素上皮细胞吞噬过程中的作用

Effect of chloride transport in phagocytic process of human retinal pigment epithelial cells

目的:探讨丁脲胺或DNDS作用下视网膜色素上皮(RPE)细胞顶端膜和基底膜上氯离子转运通道对RPE吞噬功能的影响,阐明氯离子转运通道参与PRE吞噬作用的机制。方法:利用微孔滤膜培养系统体外培养极性化单层RPE细胞,以荧光包被的乳胶株作为吞噬标记物,建立RPE细胞吞噬模型,分别以1、5、10、50、100、500和1000μmol.L-1丁脲胺,以及0.01、0.10、0.50、1.00、3.00、5.00和10.00mmol.L-1DNDS作用RPE细胞6h后,应用流式细胞仪定量检测RPE细胞吞噬指数。结果:不同浓度丁脲胺作用于RPE细胞顶端膜或基底膜,RPE细胞对乳胶株的吞噬指数与对照组比较差异无显著性(P>0.05);0.01和0.10μmol.L-1DNDS作用于RPE细胞基底膜,RPE细胞对乳胶株的吞噬指数与对照组比较差异无显著性(P>0.05),而0.50、1.00、3.00、5.00、10.00mmol.L-1的DNDS作用于RPE细胞基底膜,RPE细胞对乳胶株的吞噬指数降低(P<0.05或P<0.01),并随着浓度的增加,吞噬指数逐渐降低;但同样浓度的DNDS作用于RPE细胞顶端膜,RPE细胞对乳胶株的吞噬指数无明显改变(P>0.05)。结论:顶端膜的丁脲胺敏感性Na+-K+-2Cl-协同转运载体不参与RPE吞噬乳胶株的过程,而基底膜的DNDS敏感性Cl-/HCO3-交换蛋白及氯离子通道在DNDS作用后,对人RPE细胞非特异性吞噬过程发挥抑制作用。

Objective To investigate the effect of chloride transport in nonspecific phagocytic process of human retinal pigment epithelial （RPE） cells which was treated with DNDS and bumetanide, and to discuss the mechanism for chloride transport＇ s effort in nonspecific phagocytic process. Methods Human RPE cells （ARPE-19） were cultivated using millicell cell culture inserts system, and flow cytometry was performed on ARPE-19 cells that had been challenged with fluorescent latex beads. The bumetanide of 1, 5, 10, 50, 100, 500, 1 000 μmol · L^-1 1 and DNDS of 0.01, 0.10, 0.50, 1.00, 3.00, 5.00, 10.00 mmol· L^-1 1 were used in the experiment as chloride transport pathway blockers for 6 h, then flow cytometry was used to detect the phagocytic index. Results Compared with control group, different concentrations bumetanide didn＇t affect the nonspecific phagocytic process of RPE cells （P〉0.05） ; when the basal membrane of RPE was treated with 0.01 and 0.10 mmol · L^-1 1 DNDS, there were no significant differences of phagocytic index compared with control group （P〉0.05） ; however when treated with 0.50, 1.00, 3.00, 5.00 and 10 mmol· L^-1 1 DNDS, the phagocytic indexes were significantly decreased （P〈0.05 or P〈0.01）, and as the concentration increased, the phagocytic indexes were decreased gradually; when the apical membrane of RPE was treated with above concentrations of DNDS, there were no significant differences of phagocytic index compared with control group （P〉0.05）. Conclusion Apical membrane bumetanide-sensitive Na^＋ K^＋ -2Cl^-cotransporter doesn＇ t participate in the nonspecific phagocytic process of RPE cells, basal membrane DNDS-sensitive Cl^- /HCO3- exchange and chloride channel play important roles in the nonspecifie phagocytic process of RPE cells.