腺苷受体拮抗剂对加压培养的视网膜Müller细胞钾离子通道的调控作用  

Changes of Kir4.1 and TASK-1 expression in rat Müller cell induced by SCH442416 at an elevated hydrostatic pressure in vitro

作　　者：杨子建 程瑜 姚慧萍 沈婷 陈雁伟 钟一声 Yang Zijian;Cheng Yu;Yao Huiping;Shen Ting;Chen Yanwei;Zhong Yisheng(Department of Ophthalmology,Ruijin Hospital,Shanghai Jiao Tong University School of Medicine,Shanghai 201800,China)

机构地区：[1]上海交通大学医学院附属瑞金医院北院眼科,201800

出　　处：《中华实验眼科杂志》2018年第8期590-594,共5页Chinese Journal Of Experimental Ophthalmology

摘　　要：目的阐明腺苷受体拮抗剂SCH442416对体外加压的视网膜Müller细胞的钾离子通道的调控作用。方法出生后5 d的SPF级SD大鼠30只，将大鼠的视网膜Müller细胞分离和培养，分为正常对照组（正常培养）、加压培养组[40 mmHg/24 h加压培养（1 mmHg＝0.133 kPa）]和腺苷＋SCH442416干预组（40 mmHg/24 h加压培养后加入10 μmol/L腺苷＋100 nmol/L SCH442416培养）。对大鼠视网膜Müller细胞体外加压40 mmHg/24 h培养，采用real-time PCR、Western blot等方法研究离体加压培养的视网膜Müller细胞的钾离子通道Kir2.1、Kir4.1和TASK-1的表达变化情况。体外加压培养的视网膜Müller细胞给予腺苷＋腺苷A2A受体拮抗剂SCH442416进行干预（药物终浓度为腺苷10 μmol/L＋ SCH442416 100 nmol/L），检测视网膜及Müller细胞Kir2.1、Kir4.1和TASK-1的mRNA和蛋白的表达变化。结果40 mmHg/24 h加压培养可致视网膜Müller细胞钾离子通道Kir2.1、Kir4.1和TASK-1的蛋白表达下调，与正常对照组相比分别下降了14.7%、38.6%和52.6%，2个组Kir2.1蛋白的表达差异无统计学意义（P＝0.082），Kir4.1和TASK-1蛋白的表达差异均有统计学意义（P＝0.000、0.000）；腺苷＋ SCH442416干预组视网膜Müller细胞Kir2.1、Kir4.1和TASK-1的蛋白表达较加压培养组上调，分别上升了8.8%、60.7%和61.4%，2个组Kir2.1蛋白表达比较差异无统计学意义（P＝0.354），2个组Kir4.1和TASK-1的蛋白表达比较，差异均有统计学意义（P＝0.000、0.000）。40 mmHg/24 h加压培养可致视网膜Müller细胞Kir2.1、Kir4.1和TASK-1 mRNA表达较正常对照组下调，差异均有统计学意义（P＝0.047、0.001、0.000）；与加压培养组相比，腺苷＋ SCH442416干预组Kir4.1和TASK-1的表达均上调，差异均有统计学意义（P＝0.038、0.030），而Kir2.1的表达无明显变化，差异无统计学意义（P＝0.612）。结论SCH442416对视网膜Müller细胞Kir4.1和TASK-1的蛋白和mRNObjectiveTo evaluate the effect of adenosine receptor antagonist SCH442416 on the expression of Kir2.1, Kir4.1 and TASK-1 in rat Müller cell at an elevated hydrostatic pressure in vitro.MethodsThirty SPF Sprague Dawley rats were purchased from Shanghai Slack Laboratory Animals Ltd.Cultured Müller cells were divided into normal control group （n=6）, 40 mmHg/24 hours （1 mmHg=0.133 kPa） group （n=6） and adenosine ＋ SCH442416 intervention group（n=6）. Müller cells were treated with 40 mmHg pressure for 24 hours in 40 mmHg/24 hours group, and Müller cells were treated with 40 mmHg pressure for 24 hours ＋ 10 μmol/L adenosine ＋ 100 nmol/L SCH442416 in adenosine ＋ SCH442416 intervention group.The real-time PCR, Western blot, whole-cell patch-clamp recordings and immunohistochemistry were used to detect Kir2.1, Kir4.1 and TASK-1 expression and Müller cells Kir currents.The experimental procedures were in accordance with the National Institutes of Health （NIH） guidelines for the Care and Use of Laboratory, and follow the 3R principle. ResultsWestern blot assay showed that, following 40 mmHg pressure cultured for 24 hours, the expression of Kir4.1 and TASK-1 protein in Müller cell were significantly decreased by 38.6% and 52.6% compared with the normal control group, with significant differences between the two groups （both at P=0.000）; Kir2.1 protein expression decreased by 14.7%, with insignificant difference between the two groups （P=0.082）. Kir4.1 and TASK protein expression in adenosine ＋ SCH442416 intervention group was increased by 60.7% and 61.4% compared with the 40 mmHg/24 hours group, with significant differences between the two groups （both at P=0.000）; Kir2.1 protein expression in adenosine ＋ SCH442416 intervention group was increased by 8.8% compared with the 40 mmHg/24 hours group, with insignificant difference between them （P=0.354）. Real-time PCR assay showed that, following 40 mmHg pressure cultured for 24 hours, Kir2.1, Kir4.1 and TASK-1 mRNA expression

关 键 词：视网膜 MÜLLER细胞 SCH442416 KIR2.1 Kir4.1 TASK-1 

分 类 号：R775[医药卫生—眼科]