细胞外钙离子内流上调原发性开角型青光眼患者小梁网细胞Copine1表达的研究

Effect of influx of Ca^(2+) on the expression of copine 1 in human trabecular meshwork cells in primary open angle glaucoma

目的探讨原发性开角型青光眼患者(primary open-angle glaucoma,POAG)小梁网细胞外钙离子内流对膜蛋白copine1表达的影响。方法原代培养POAG患者小梁网细胞,应用荧光显微镜和流式细胞仪检测细胞内钙离子浓度,所用钙离子荧光探针为Fluo 3-AM;;应用钙离子运载剂A23187增加细胞内钙离子浓度;;应用Real-time PCR检测小梁网细胞内钙离子浓度增加前后copine1在转录水平的表达。结果经免疫组织化学鉴定培养的细胞为小梁网细胞。荧光显微镜下观察与流式细胞仪定量结果均显示,应用钙离子运载剂A23187增加了POAG患者小梁网细胞内的钙离子浓度。Real-time PCR检测证实A23187的应用使copine1 mRNA在小梁网细胞中表达增加了(1.8±0.3)倍。结论 POAG患者小梁网细胞内钙离子浓度增高可引起copine1在转录水平表达的上调,copine1在小梁网细胞中的功能研究将会增加对POAG病理学机制的理解,有助于新药物靶点的开发。

Objective To investigate the effect of influx of Ca2＋ on the expres- sion of copine 1 in human trabecular meshwork（TM） cells in primary open angle glau- coma（POAG）. Methods TM cells in POAG patients were primarily cultured. The in- tracellular calcium concentration（ Ca2 ＋ ） was measured by the fluorescence microscope and flow cytometery,in which fluorescent probe Fluo3-AM was used. The Ca2 ＋ in TM cells was increased by incubating with calcium ionophore,A23187. The expression levels of copinel were measured both in untreated and A23187-treated TM cells by real-time polymerase chain reaction（PCR）. Results The cells cultured by immunohistochemis- try were TM cells. Both the results of fluorescence microscope and flow cytometery showed that the incubating with A23187 increased the Ca2＋ of TM cells in POAG pa- tients. Real-time PCR confirmed that copinel mRNA expression was（ 1.8 ± 0.3 ） times of the untreated group after incubating with A23187. Conclusion The up-regulation of copinel in TM cells relates to the increace of POAG,which may contribute to the un- derstanding of pathology mechanism of POAG and development of new drug target.