Background Conjunctivochalasis (CCh) is a common age-related ocular surface diseases.Researches showed that the inflammatory factors are upregulated in conjunctiva and tear of CCh patients,inferring inflammation participates in the pathogenesis of CCh,however,its mechanism is unclear now.Studies also showed that the expression of matrix metalloproteinases (MMPs) in CCh conjunctiva is elevated.However,the association between inflammatory factors and MMPs is unknown.Objective This research was to observe the effects of tumor necrosisfactor-α (TNF-α) and interleukin-1 β (IL-1 β) on the expression of MMP-1,MMP-3,tumor necrosis factor-stimulatedgene-6(TSG-6) and pentraxin-3 (PTX3) in cultured CCh fibroblasts.Methods Conjunctival fibroblasts wereisolated and cultured from CCh-derived and cataract-derived human conjunctiva explants.The cells were treated using20 ng/ml PBS,TNF-α or IL-1β for 4 hours,respectively.The expression levels of MMP-1,MMP-3,TSG-6 and PTX3genes and proteins were detected by quantitative real time PCR and Western blot assay,respectively.Results Thesecond-generation cells showed a long and fusiform shape with ovoid nucleus and radial agreement,and the cellprocessors connected each other.The differences of the relative expression levels of MMP-1,MMP-3,TSG-6 and PTX3mRNA in the cells were significantly different between CCh group and cataract group after being treated by PBS,TNF-αand IL-1 β (MMP-1 mRNA:Fgroup =2.611,P =0.116;Fi =161.564,P =0.000;MMP-3 mRNA:Fgroup =5.201,P =0.029;Fintervene =211.021,P =0.000;TSG-6 mRNA:Fgroup =47.209,P =0.000;Fintervene =119.340,P =0.000;PTX3 mRNA:Fgroup =40.512,P =0.000;Fintervene =93.935,P =0.000).Compared with the cataract group with PBS treatment,the expression levels of MMP-1,MMP-3,TSG-6 and PTX3 mRNA were significantly elevated in the CCh group with PBS treatment or the cataract group with TNF-α and IL-1 β treatment (all at P＜0.05).Compared with the CCh group with PBS treatment,the expression levels of MMP-1,MMP-3,TSG-6 and PTX3 mRNA were significantly elevated in the CCh group with TNF-α and IL-1β treatment (all at P ＜ 0.05).The differences of the relative expression levels of MMP-1,MMP-3,TSG-6 and PTX3 proteins in the cells were significantly different between CCh group and cataract group after being treated by PBS,TNF-α and IL-1 β (MMP-1:Fgroup =84.702,P =0.000;Fint =48.900,P =0.000;MMP-3:Fgroup =112.818,P =0.000;Fint =194.980,P =0.000;TSG-6:Fgroup =56.867,P =0.000;Fint =70.356,P =0.000;PTX3:Fgroup =1.488,P =0.231;Fint =89.872,P =0.000),and the expression changes of MMP-1,MMP-3,TSG-6 and PTX3 proteins were coincident with the genes among the groups with various treatments.Conclusions The expressions of MMP-1,MMP-3,TSG-6 and PTX3 in conjunctival fibroblasts are upregulated in CCh eyes.The interaction of TNF-α and IL-1 β with MMPs is probably involved with the pathogenesis and development MMPs probably.%背景 结膜松弛症(CCh)是年龄相关性眼表疾病,研究发现CCh患者结膜组织及泪液中炎性细胞因子表达升高,推测炎性细胞因子可能参与CCh的发病,但其作用机制尚未完全阐明.研究还发现CCh患者结膜组织中基质金属蛋白酶(MMPs)的表达上调,而炎性因子与MMPs在CCh发病中是否有关联尚不清楚. 目的 探讨肿瘤坏死因子α(TNF-α)和白细胞介素1β(IL-1β)对CCh患者结膜成纤维细胞中MMP-1、MMP-3及TNF-α刺激产生的肿瘤坏死因子刺激基因6(TSG-6)和穿透素3(PTX3)表达的影响.方法 采用组织块培养法分别对CCh和白内障患者手术中采集的结膜组织进行培养,将培养的2种结膜成纤维组织各分为3个组,分别用含20 ng/ml的PBS、TNF-α或IL-1β的细胞全培养基培养细胞4h,分别采用实时荧光定量PCR法和Western blot法检测细胞中MMP-1、MMP-3、TSG-6和PTX3 mRNA及其蛋白的表达.结果 倒置显微镜下可见第2代细胞的形态和大小一致,为长梭形,呈放射状排列,细胞核呈卵圆形,细胞突起相互交联.CCh组和白内障组间用PBS、TNF-α和IL-1β干预后细胞中MMP-1、MMP-3、TSG-6和PTX3 mRNA相对表达量的总体比较差异均有统计学意义(MMP-1 mRNA:F分组=2.611,P=0.116;F干预=161.564,P=0.000;MMP-3mRNA:F分组=5.201,P=0.029;F干预=211.021,P=0.000;TSG-6 mRNA:F分组=47.209,P=0.000;F干预=119.340,P=0.000;PTX3 mRNA:F分组=40.512,P=0.000;F干预=93.935,P=0.000).与白内障组PBS干预后比较,CCh组PBS干预后MMP-1、MMP-3、TSG-6和PTX3 mRNA的相对表达量均明显升高,差异均有统计学意义(均P＜0.05);与CCh组PBS干预后比较,CCh组TNF-α和IL-1β干预后细胞中MMP-1、MMP-3、TSG-6和PTX3 mRNA的相对表达量明显升高,差异均有统计学意义(均P＜0.05);与白内障组PBS干预后比较,白内障组TNF-α和IL-1β干预后细胞中MMP-1、MMP-3、TSG-6和PTX3 mRNA相对表达量明显增高,差异均有统计学意义(均P＜0.05).CCh组和白内障组间用PBS、TNF-α和IL-1β干预后细胞中MMP-1、MMP-3、TSG-6和PTX3蛋白相对表达量的总体比较差异均有统计学意义(MMP-1:F分组=84.702,P=0.000;F干预=48.900,P=0.000;MMP-3:F分组=112.818,P=0.000;F干预=194.980,P=0.000;TSG-6:F分组=56.867,P=0.000;F干预=70.356,P=0.000;PTX3:F分组=1.488,P=0.231;F干预=89.872,P=0.000).各组细胞用PBS、TNF-α和IL-1β干预后MMP-1、MMP-3、TSG-6和PTX3蛋白相对表达量的组间比较与其基因表达趋势一致. 结论 CCh患者结膜成纤维细胞中MMP-1、MMP-3、TSG-6和PTX3的表达量增加;TNF-α和IL-1β均可上调CCh结膜成纤维细胞中MMP-1、MMP-3、TSG-6和PTX3的表达.
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