基质金属蛋白-9抑制剂阻断视网膜色素上皮细胞表面CD73脱离防治实验性自身免疫性葡萄膜炎的初步研究

Q4 Medicine

中华眼底病杂志 Pub Date : 2020-04-25 DOI:10.3760/CMA.J.CN511434-20180502-00141

Shumin Zhou, Fanqiang Kong, Song Chen

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The cells in each group were treated with the intervention of a solvent, 1 μmol/Ladenosine monophosphate (AMP), 1 μmol/L AMP, and 3 μmol/L 5' -α,β-methylene adenosine diphosphate (APCP) (AMP+APCP). The stimulating effect of RPE cells in different groups on CD4+ T cell proliferation was detected by tritiated thymidine incorporation. Adoptive immune induced EAU in wild-type B6 mice and CD73-/- mice, respectively. The receptor mice were randomly divided into the MMP-9 inhibitor intervention group and the non-intervention control group, and CTK8G1150 or the solvent were injected into the subretinal cavity 4, 7 and 10 days after adoptive immunity. CD73 mRNA and protein expression in RPE cells of recipient mice were detected by real-time quantitative PCR (RT-PCR) and Western blot. 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引用次数: 0

摘要

目的初步探讨基质金属蛋白酶9阻断CD73从RPE细胞表面脱离，防治实验性自身免疫性色素膜炎（EAU）的机制。方法体外培养野生型C57BL/6和CD73基因敲除（CD73-/-）小鼠RPE细胞，并用脂多糖和TNF-α处理以诱导CD73从RPE表面脱离。根据是否同时加入MMP-9抑制剂CTK8G1150（终浓度为5.0mol/L），将两种小鼠培养的RPE细胞分别设为MMP-9抑制剂干预组和非干预对照组。每组细胞用溶剂1μmol/一磷酸拉地诺辛（AMP）、1μmol/L AMP和3μmol/L 5’-α，β-亚甲基腺苷二磷酸（APCP）（AMP+ACP）干预。用氚化胸苷掺入法检测不同组RPE细胞对CD4+T细胞增殖的刺激作用。过继免疫分别在野生型B6小鼠和CD73-/-小鼠中诱导EAU。受体小鼠被随机分为MMP-9抑制剂干预组和非干预对照组，并在过继免疫后4、7和10天将CTK8G1150或溶剂注射到视网膜下腔。采用实时定量PCR（RT-PCR）和蛋白质印迹法检测受体小鼠RPE细胞中CD73 mRNA和蛋白的表达。单因素方差分析用于分析所有实验数据。结果当刺激方式为AMP时，与未干预组相比，C57BL/6 MMP-9抑制剂干预组CD4+T细胞的增殖显著降低（F=13.28，P 0.05）。CD73-/-MMMP-9抑制剂介入组CD4+T细胞的增殖能力与无干预组相比无统计学意义（F=5.24，6.12，7.04；P＞0.05）。RT-PCR结果结果显示，MMP-9抑制剂组RPE细胞中CD73 mRNA的相对表达与未干预对照组相比无统计学意义（F=6.54，P>0.05）（F=15.24，P<0.01）。结论MMP-9抑制剂阻断CD73从RPE细胞表面脱离，对EAU有保护作用。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；细胞，培养；葡萄膜炎/生理病理学；动物实验

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Preliminary study on prevention and treatment of experimental autoimmune uveitis by blocking CD73 detachment from the surface of retinal pigment epithelial cells with matrix metalloprotein-9 inhibitor

Objective To preliminarily investigate the mechanism of MMP-9 blocking CD73 detachment from RPE cells surface and preventing and treating experimental autoimmune pigment membranitis(EAU). Methods RPE cells isolated from wild-type C57BL/6 and CD73 gene knockout (CD73-/-) mice were cultured in vitro, and treated with lipopolysaccharide and TNF-α to induce CD73 detachment from RPE surface. According to whether MMP-9 inhibitor CTK8G1150 was added at the same time (the final concentration was 5.0 mol/L) or not, RPE cells cultured in the two types of mice were respectively set as MMP-9 inhibitor intervention group and non-intervention control group. The cells in each group were treated with the intervention of a solvent, 1 μmol/Ladenosine monophosphate (AMP), 1 μmol/L AMP, and 3 μmol/L 5' -α,β-methylene adenosine diphosphate (APCP) (AMP+APCP). The stimulating effect of RPE cells in different groups on CD4+ T cell proliferation was detected by tritiated thymidine incorporation. Adoptive immune induced EAU in wild-type B6 mice and CD73-/- mice, respectively. The receptor mice were randomly divided into the MMP-9 inhibitor intervention group and the non-intervention control group, and CTK8G1150 or the solvent were injected into the subretinal cavity 4, 7 and 10 days after adoptive immunity. CD73 mRNA and protein expression in RPE cells of recipient mice were detected by real-time quantitative PCR (RT-PCR) and Western blot. One-way ANOVA was used to analyze all experimental data. Results When the stimulation mode was AMP, the proliferation of CD4+ T cells in the C57BL/6 MMP-9 inhibitor intervention group decreased significantly compared with the nonintervention group (F=13.28, P 0.05). There was no statistically significant difference in the proliferation capacity of CD4+ T cells between the CD73-/- MMP-9 inhibitor intervention group and the non-intervention group (F=5.24, 6.12, 7.04; P>0.05). RT-PCR results showed that there was no statistically significant difference in the relative expression of CD73 mRNA in RPE cells between the MMP-9 inhibitor group and the non-intervention control group(F=6.54, P>0.05). Western blot results showed that the expression of CD73 protein in RPE cells in the MMP-9 inhibitor group of B6 receptor mice was significantly increased compared with the control group (F=15.24, P<0.01). Conclusion MMP-9 inhibitor blocks CD73 detachment from RPE cells surface and has a protective effect on EAU. Key words: Matrix metalloproteinase 9/antagonists & inhibitors; 5'-nucleotidase; Retinal pigment epithelium; Cells, cultured; Uveitis/physiopathology; Animal experimentation

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来源期刊

中华眼底病杂志 Medicine-Ophthalmology

CiteScore

0.40

自引率

0.00%

发文量

5383

期刊介绍： Chinese Journal of Ocular Fundus Diseases is the only scientific journal in my country that focuses on reporting fundus diseases. Its purpose is to combine clinical and basic research, and to give equal importance to improvement and popularization. It comprehensively reflects the leading clinical and basic research results of fundus disease disciplines in my country; cultivates professional talents in fundus disease, promotes the development of fundus disease disciplines in my country; and promotes academic exchanges on fundus disease at home and abroad. The coverage includes clinical and basic research results of posterior segment diseases such as retina, uveal tract, vitreous body, visual pathway, and internal eye diseases related to systemic diseases. The readers are medical workers and researchers related to clinical and basic research of fundus diseases. According to the journal retrieval report of the Chinese Institute of Scientific and Technological Information, the comprehensive ranking impact factor and total citation frequency of the Chinese Journal of Ocular Fundus Diseases have been among the best in the disciplines of ophthalmology, otolaryngology, and ophthalmology in my country for many years. The papers published have been included in many important databases at home and abroad, such as Scopus, Peking University Core, and China Science Citation Database (CSCD).