缓激肽刺激体外培养的视网膜色素上皮细胞炎症反应的作用机制

摘要

Abstract?AIM: To investigate mechanism of bradykinin ( BK) on inflammations of retinal pigment epithelium ( RPE) cells.?METHODS: ARPE -19 cells were cultured in vitro, stimulated by 100nM BK for 24h. Cell morphology changes were observed by microscope, and BK receptor localization was detected through cell immunofluorescence. Changes of Ca2+in BK and BR antagonist stimuli were detected by laser scanning confocal microscopy.The expressions of COX-1, COX-2, eNOS and iNOS protein in control group and BK group were detected by Western Blot.?RESULTS: After the stimulation of BK, there was no significant changes of ARPE-19 cells in morphology.Kinin B1 receptors ( B1R ) and B2 receptors ( B2R ) could be detected in ARPE-19 cells.Compared with control group, Ca2+concentrations significantly increased in BK group; in B1R antagonist group and B2R antagonist group Ca2+concentrations increased less than BK group; B1R and B2R antagonist group showed no obvious changes in Ca2+concentrations.Compared with control group, COX-2 and iNOS protein concentrations were significantly increased in BK group (P<0.001).?CONCLUSION:BK induces the increasing expression of COX-2 and iNOS in the cultured ARPE cells through binding with either B1R or B2R.%目的：探讨缓激肽刺激体外培养的视网膜色素上皮（ retinal pigment epithelium ， RPE ）细胞炎症反应的作用机制。方法：体外培养 ARPE－19细胞，100 nmol／L 缓激肽（bradykinin，BK）刺激24h后，光镜下观察细胞形态变化，细胞免疫荧光检测BK受体定位；共聚焦显微镜检测BK及其拮抗剂作用下Ca2＋变化；Western blot 检测对照组与100 nmol／L BK 处理24 h 后（ BK 组） COX－1、COX－2、eNOS、iNOS蛋白的表达量。结果：BK刺激后，ARPE－19细胞形态无明显变化；ARPE－19细胞可检测到激肽B1、B2受体；与对照组相比， BK组Ca2＋浓度显著升高；B1R拮抗组及B2R拮抗组的Ca2＋浓度较BK组升高幅度降低，B1R及B2R拮抗组Ca2＋浓度较对照组无明显变化；BK作用ARPE－19细胞后， COX－2及iNOS蛋白含量显著增加（P＜0．001）。结论：BK通过与B1 R及B2 R结合促进体外培养的ARPE细胞COX－2及iNOS表达增加。