中华眼底病杂志

中华眼底病杂志

基质金属蛋白酶-9调控CD73自视网膜色素上皮细胞膜表面脱落机制的初步研究

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目的探讨基质金属蛋白酶(MMP)-9调控CD73自视网膜色素上皮(RPE)细胞膜表面脱落的机制。方法以脂多糖及肿瘤坏死因子-α共同干预体外培养的人RPE细胞,诱导RPE细胞膜表面CD73脱落。同时给予磷脂酶C(PLC)抑制剂ET-18-OCH3 30.0 μmol/L或广谱MMP抑制剂ONO-4817 5.0 μmol/L进行干预;流式细胞仪检测RPE细胞膜表面CD73含量变化。以选择性MMP-2、MMP-9抑制剂或相应siRNA进行干预;利用基因定点突变技术改造CD73中潜在的MMP-9识别位点,在CD73-/- RPE细胞中过表达野生型(Wt)及突变型(Mut)CD73,检测Wt、Mut-CD73脱落情况。结果广谱MMP抑制剂可有效抑制CD73自RPE细胞膜表面脱落;PLC抑制剂不能抑制CD73自RPE细胞膜表面脱落。选择性抑制MMP-9活性或以siRNA下调MMP-9的表达也均可抑制CD73自RPE细胞膜表面脱落。表达于CD73-/-RPE细胞膜表面的Wt-CD73可发生脱落,而Lys547-Phe548编码位点突变的CD73未见脱落。结论MMP-9通过水解Lys547-Phe548位点而参与炎症状态下CD73自RPE细胞膜表面的脱落。

ObjectiveTo study how CD73 is shed from the retinal pigment epithelium (RPE) surface.MethodsCD73 shedding was induced by treating RPE with lipopolysaccharides (LPS) and TNF-α. After Phospholipase C (PLC) or pan matrix metalloproteinase (MMP) inhibitors were added, surface amount of CD73 was evaluated by flow cytometry (FACS). Then selective inhibitors or their corresponding siRNAs of MMP-2 and MMP-9 were applied to the treatments of RPE; and their effects on induced CD73 shedding were evaluated by FACS. By site directed mutagenesis, mutations were introduced to Lys547-Phe548 coding sites of CD73 cDNA, which was cloned in a pcDNA mammalian expression vector. Both wt-CD73 and mutated-CD73 were over expressed in CD73-/- RPE and their induced shedding was compared.ResultsLPS and TNF-α induced CD73 shedding from RPE was completely blocked by the addition of pan MMP inhibitor but not PLC inhibitor. Selective MPP-9, but not MMP-2, inhibitor or its siRNA blocked CD73 shedding. In CD73-/- RPE induced CD73 shedding was happened to overexpressed wt-CD73 but not Lys547-Phe548 sites mutant CD73.ConclusionMMP-9 is responsible for shedding CD73 from RPE through hydrolyzing its Lys547 -Phe548 sites.

关键词: 基质金属蛋白酶-9/拮抗剂和抑制剂; 5'-核苷酸酶; 视网膜色素上皮

Key words: Matrix metalloproteinase 9/antagonists & inhibitors; 5'-Nucleotidase; Retinal pigment epithelium

引用本文: 孔繁强, 周树民, 陈松. 基质金属蛋白酶-9调控CD73自视网膜色素上皮细胞膜表面脱落机制的初步研究. 中华眼底病杂志, 2017, 33(5): 518-522. doi: 10.3760/cma.j.issn.1005-1015.2017.05.018 复制

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