Chen Lei, Liu Gao-Qin, Wu Hong-Ya, Jin Ji, Yin Xue, Li Dan, Lu Pei-Rong
Department of Ophthalmology, the First Affiliated Hospital of Soochow University, Suzhou 215006, Jiangsu Province, China.
Jiangsu Key Laboratory of Clinical Immunology, Soochow University, Suzhou 215006, Jiangsu Province, China.
Int J Ophthalmol. 2018 Feb 18;11(2):216-222. doi: 10.18240/ijo.2018.02.06. eCollection 2018.
To explore the interaction between macrophages and chemokines [monocyte chemoattractant protein 1 (MCP-1/CCL2) and fractalkine/CX3CL1] and the effects of their interaction on neovascularization.
Human peripheral blood mononuclear cells, donated by healthy volunteers, were separated and cultured in RPMI-1640 medium containing 10% fetal bovine serum, then induced into macrophages by stimulation with 30 µg/L granulocyte macrophage-colony stimulating factor (GM-CSF). The expression of CCR2 and/or CX3CR1 in the macrophages was examined using flow cytometry. Macrophages were then stimulated with recombinant human CCL2 (rh-CCL2) or recombinant human CX3CL1 (rh-CX3CL1). The expression of angiogenesis-related genes, including , and were examined using real-time quantitative polymerase chain reaction (PCR). Supernatants from stimulated macrophages were used in an assay of human retinal endothelial cell (HREC) proliferation. Finally, stimulated macrophages were co-cultured with HREC in a migration assay.
The expression rate of CCR2 in macrophages stimulated by GM-CSF was 42%±1.9%. The expression rate of CX3CR1 was 71%±3.3%. Compared with vehicle-treated groups, gene expression of in the macrophages was greater in 150 mg/L CCL2-treated groups (<0.05), while expression of and was significantly lower (<0.05). By contrast, compared with vehicle-treated groups, expression of in 150 mg/L CX3CL1-treated groups was significantly lower (<0.05), while expression of and was greater (<0.05). Supernatants from CCL2 treated macrophages promoted proliferation of HREC (<0.05), while supernatants from CX3CL1-treated macrophages inhibited the proliferation of HREC (<0.05). HREC migration increased when co-cultured with CCL2-treated macrophages, but decreased with CX3CL1-treated macrophages (<0.05).
CCL2 and CX3CL1 exert different effects in regulation of macrophage in expression of angiogenesis-related factors, including , and . Our findings suggest that CCL2 and CX3CL1 may be candidate proteins for further exploration of novel targets for treatment of ocular neovascularization.
探讨巨噬细胞与趋化因子[单核细胞趋化蛋白1（MCP-1/CCL2）和 fractalkine/CX3CL1]之间的相互作用及其相互作用对新生血管形成的影响。
分离健康志愿者捐献的人外周血单个核细胞，在含10%胎牛血清的RPMI-1640培养基中培养，然后用30μg/L粒细胞巨噬细胞集落刺激因子（GM-CSF）刺激诱导为巨噬细胞。采用流式细胞术检测巨噬细胞中CCR2和/或CX3CR1的表达。然后用重组人CCL2（rh-CCL2）或重组人CX3CL1（rh-CX3CL1）刺激巨噬细胞。采用实时定量聚合酶链反应（PCR）检测血管生成相关基因，包括 、 和 的表达。将刺激后的巨噬细胞培养上清用于人视网膜内皮细胞（HREC）增殖检测。最后，将刺激后的巨噬细胞与HREC共培养进行迁移检测。
GM-CSF刺激的巨噬细胞中CCR2的表达率为42%±1.9%。CX3CR1的表达率为71%±3.3%。与溶剂处理组相比，150mg/L CCL2处理组巨噬细胞中 的基因表达更高（<0.05），而 和 的表达显著更低（<0.05）。相比之下，与溶剂处理组相比，150mg/L CX3CL1处理组中 的表达显著更低（<0.05），而 和 的表达更高（<0.05）。CCL2处理的巨噬细胞培养上清促进HREC增殖（<0.05），而CX3CL1处理的巨噬细胞培养上清抑制HREC增殖（<0.05）。与CCL2处理的巨噬细胞共培养时HREC迁移增加，但与CX3CL1处理的巨噬细胞共培养时迁移减少（<0.05）。
CCL2和CX3CL1在调节巨噬细胞血管生成相关因子 、 和 的表达方面发挥不同作用。我们的研究结果表明，CCL2和CX3CL可能是进一步探索治疗眼部新生血管新靶点的候选蛋白。
