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

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（5 μmol/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）10 μg/L 及 γ 干扰素（IFN-γ）1 000 U/mL 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。

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

Objective To clarify which adenosine receptor subtype is the most powerful one on controlling retinal pigment epithelial cell (RPE) binding adenosine, and what is its function in RPE. Methods Total mRNA was isolated, and membrane protein was extracted from in vitro cultured human ARPE-19 cells. For all four kinds of adenosine receptors, ARA1, ARA2A, ARA2B and ARA3, their gene expressions were tested by real-time PCR while their molecules in the membrane protein were detected by Western blot assay. To check the influence of each adenosine receptor subtype on ARPE-19 cell binging ability to adenosine the cultured cells were divided into five groups, named A-E. A group was set up as untreated control, while, groups B-E were separately treated by ARA1 agonist DPCPX (50 nmol / L), ARA2A agonist SCH58261 (100 nmol/L), ARA2B agonist MRS1754 (100 nmol/L) or ARA3 agonist MRS1220 (5 μmol/L). H3-adenosine a radioactive ligand binding assay was performed and the maximum binding capacities (Bmax) were calculated in groups A-E of ARPE-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. Results Either mRNA expression or membrane localization of ARA1, ARA2A, ARA2B and ARA3 were verified by realtime 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 in group 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 significant difference in TGF- β content between the two groups. Conclusion APRE-19 cells predominantly use ARA1 to absorb adenosine, and the activation of ARA1 in ARPE-19 cells inhibits its IL-6, MCP-1, and IP-10 production, which have potentially immunosuppressive effects to APRE-19 cells.