腺苷受体A1亚型对视网膜色素上皮细胞免疫调节功能的影响  

The influence of adenosine receptor A1 subtype on the immune regulatory function of retinal pigment epithelium cells

作　　者：孔繁强[1] 周树民[2] 张伟[3] 陈松[4] 

机构地区：[1]天津医科大学总医院医学检验科,300052 [2]天津医科大学第二医院检验科 [3]天津市眼科医院斜视科 [4]天津医科大学总医院眼科

出　　处：《天津医药》2018年第1期12-15,共4页Tianjin Medical Journal

基　　金：国家自然科学基金资助项目(81570833)

摘　　要：目的研究视网膜色素上皮细胞(RPE)主要是通过何种亚型的腺苷受体(ARs)来结合腺苷,及其对RPE功能的影响。方法体外培养人ARPE-19细胞系,定量PCR检测4种腺苷受体(ARA1、ARA2A、ARA2B、ARA3)基因的表达;提取细胞膜蛋白,Western blot检测4种腺苷受体在RPE细胞膜上的存在。体外培养ARPE-19细胞至80%融合后随机分为A^E组。其中,A组为无干预对照组,B^E组分别给予ARA1拮抗剂DPCPX(50 nmol/L)、ARA2A拮抗剂SCH58261(100 nmol/L)、ARA2B拮抗剂MRS1754(100 nmol/L)及ARA3拮抗剂MRS1220(5μmol/L)干预。利用H3-腺苷进行放射性配体结合实验,计算各组细胞对腺苷的最大结合容量(Bmax)。以肿瘤坏死因子α(TNF-α)10μg/L及γ干扰素(IFN-γ)1 000 U/m L联合干预体外培养的ARPE-19细胞,给予或不予ARA1激动剂(CCPA),酶联免疫吸附试验(ELISA)测定培养上清中白细胞介素(IL)-6、IL-10、转化生长因子β(TGF-β)、单核细胞趋化因子(MCP)-1、趋化因子C-X-C配体10(CXCL10,IP-10)的含量。结果在ARPE-19细胞中即可检测到4种腺苷受体基因的表达,也可探测到其分子在细胞膜上的存在。A^E组ARPE-19细胞结合腺苷的Bmax(单位:fmol)分别为2.04±0.31、0.44±0.06、1.82±0.28、2.01±0.42及2.06±0.44,其中B组较其他各组Bmax均降低(P<0.01)。以TNF-α及IFN-γ激活ARPE-19细胞,与对照RPE组比较,CCPA干预RPE组IL-6、MCP-1及IP-10的含量降低、IL-10的含量增加(P<0.01)。2组TGF-β的含量差异无统计学意义。结论 ARA1对ARPE-19细胞结合腺苷的能力具有重要的调控作用,ARA1受体介导的信号可抑制ARPE-19细胞分泌促炎因子及驱化因子,具有潜在的免疫抑制作用。Objective To clarify which adenosine receptor subtype is the most powerful one on controlling retinalpigment epithelial cell(RPE) binding adenosine, and what is its function in RPE.MethodsTotal m RNA was isolated, andmembrane protein was extracted from in vitro cultured human ARPE-19 cells. For all four kinds of adenosine receptors,ARA1, ARA2 A, ARA2 B and ARA3, their gene expressions were tested by real-time PCR while their molecules in themembrane protein were detected by Western blot assay. To check the influence of each adenosine receptor subtype onARPE-19 cell binging ability to adenosine the cultured cells were divided into five groups, named A-E. A group was set upas untreated control, while, groups B-E were separately treated by ARA1 agonist DPCPX(50 nmol/L), ARA2 A agonistSCH58261(100 nmol/L), ARA2 B agonist MRS1754(100 nmol/L) or ARA3 agonist MRS1220(5 μmol/L). H3-adenosine aradioactive ligand binding assay was performed and the maximum binding capacities(Bmax) were calculated in groups A-E ofARPE-19 cells. Then, ARPE-19 cells were all treated by the combination of TNF-α and IFN-γ but with or without CCPA(100 nmol/L), an ARA1 agonist. MCP-1, IP-10, IL-6, IL-10 and TGF-β in their mediums were determined by ELISA.ResultsEither m RNA expression or membrane localization of ARA1, ARA2 A, ARA2 B and ARA3 were verified by real-time PCR and Western blot assay respectively. For A-E groups of ARPE-19 cells the Bmax of adenosine binding were(2.04±0.31),(0.44±0.06),(1.82±0.28),(2.01±0.42) and(2.06±0.44) fmol respectively; and which were statistically decreased ingroup B than those of all other groups(P<0.01). Compared with control RPE, the contents of IL-6, MCP-1 and IP-10 were decreased after treatment with CCPA, and the content of IL-10 increased in RPE group(P<0.01). There was no significantdifference in TGF-β content between the two groups.ConclusionAPRE-19 cells predominantly use ARA1 to absorbadenosine, and the activation of ARA1 in ARPE-19 cells inhibits its IL-6, MCP-1, and IP-10 production, which

关 键 词：受体 嘌呤能P1 视网膜色素上皮细胞 结合 功能 A1亚型 

分 类 号：R773[医药卫生—眼科]