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

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

作　　者：周树民[1] 孔繁强[2] 陈松[3] Zhou Shumin;Kong Fanqiang;Chen Song(The Second Hospital of Tianjin Medical University,Tianjin 300211,China;Laboratory Medicine,General Hospital of Tianjin Medical University,Tianjin 300052,China;Department of Ophthalmology,General Hospital of Tianjin Medical University,Tianjin 300052,China)

机构地区：[1]天津医科大学第二医院检验科,300211 [2]天津医科大学总医院检验科,300052 [3]天津医科大学总医院眼科,300052

出　　处：《中华眼底病杂志》2020年第4期289-294,共6页Chinese Journal of Ocular Fundus Diseases

基　　金：国家自然科学基金(81570833)。

摘　　要：目的:初步探讨MMP-9抑制剂阻断RPE细胞表面CD73脱落,防治实验性自身免疫葡萄膜炎(EAU)的机制。方法:体外培养分离自野生型C57BL/6小鼠及CD73基因敲除(CD73-/-)小鼠的RPE细胞,以脂多糖及TNF-α共同干预,诱导RPE表面CD73脱落。依据是否同时加入MMP-9抑制剂CTK8G1150(终浓度5.0μmol/L),将两种小鼠培养的RPE细胞分别设为MMP-9抑制剂干预组及无干预对照组。每组细胞给予溶媒、1μmol/L单磷酸腺苷(AMP)、1μmol/L AMP及3μmol/L 5'-α,β-亚甲基-二磷酸腺苷(APCP)(AMP+APCP)进行干预。氚化胸苷掺入法检测不同组别RPE细胞对CD4+T细胞增生的刺激作用。分别以野生型B6小鼠及CD73-/-小鼠为受体,过继免疫诱发EAU。受体小鼠分别随机分为MMP-9抑制剂干预组及未干预对照组,于过继免疫后4、7、10 d视网膜下腔分别注射CTK8G1150或溶媒。实时定量PCR(RT-PCR)、Western blot法检测受体小鼠RPE细胞中CD73 mRNA及蛋白表达。组间比较采用单因素方差分析。结果:刺激方式为AMP时,C57BL/6 MMP-9抑制剂干预组CD4+T细胞的增生能力较无干预组显著下降,差异有统计学意义(F=13.28,P<0.01);溶媒、AMP+APCP时,两组CD4+T细胞增生能力比较,差异无统计学意义(F=7.78、6.58,P>0.05)。CD73-/-MMP-9抑制剂干预组、无干预组不同刺激方式CD4+T细胞增生能力比较,差异均无统计学意义(F=5.24、6.12、7.04,P>0.05)。RT-PCR检测结果显示,B6受体小鼠MMP-9抑制剂组、无干预对照组RPE细胞中CD73 mRNA相对表达量比较,差异无统计学意义(F=6.54,P>0.05)。Western blot检测结果显示,B6受体小鼠MMP-9抑制剂组RPE细胞中CD73蛋白表达较未干预对照组显著增加,差异有统计学意义(F=15.24,P<0.01)。结论:MMP-9抑制剂阻断RPE细胞表面CD73的脱落对EAU具有保护作用。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-αto 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,1μmol/Ladenosine monophosphate(AMP),1μmol/L AMP,and 3μmol/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<0.01).When the stimulation mode was solvent and AMP+APCP,there was no statistically significant difference in the proliferation capacity of CD4+T cells between the two groups(F=7.78,6.58;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

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 培养的 葡萄膜炎/病理生理学 动物实验 

分 类 号：R77[医药卫生—眼科]