抑肽酶对翼状胬肉成纤维细胞肿瘤坏死因子-α的抑制作用

Aprotinin inhibits TNF-α expression induced by hydrogen peroxide in fibroblasts

目的探讨抑肽酶对由过氧化氢诱导的体外培养的翼状胬肉成纤维细胞存活率及分泌肿瘤坏死因子-α(tumornecrosis factor-α,TNF-α)的影响。方法翼状胬肉成纤维细胞原代、传代培养,取第3到第5代细胞做实验。本实验分5组,以不加抑肽酶为对照组,实验组分别加入1500U/ml、3000U/ml、6000U/ml、12000U/ml的抑肽酶,且每组都用过氧化氢诱导,根据作用时间的不同,再分为不同的亚组,分别作用15min、30min、60min、6h、24h。以四甲基偶氮唑盐(MTT)法检测不同质量浓度抑肽酶对细胞存活率的影响;应用ELISA法检测用不同浓度的抑肽酶作用24h后上清液中TNF-α的含量。结果ELISA法显示只有当抑肽酶浓度为1500U/ml、3000U/ml时能够明显地抑制过氧化氢诱导的翼状胬肉成纤维细胞分泌的TNF-α的量,在过氧化氢作用30min、60min、6h、24h时,其抑制量分别为19.36pg/ml、38.46pg/ml、56.15pg/ml、13.84pg/ml和10.2pg/ml、31.46pg/ml、49.15pg/ml、4.62pg/ml(P<0.05);且在该浓度时MTT法显示抑肽酶对细胞增殖无促进作用。结论抑肽酶可有效地抑制翼状胬肉成纤维细胞TNF-α的分泌而无促进增殖作用。

Objective To study the antiproliferation effect of aprotinin and the antiexpression effect of TNF-α on pterygium fibroblasts in vitro. This may indentify a potential agent for the prevention and treatment of primary or recurring pterygium.Methods First, a primary culture and subculture of pterygium fibroblasts were established in vitro. Hydrogen peroxide （150 μmol/L） was used to stimulate TNF-α expression in pterygium fibroblasts at 15, 30 and 60 minutes and 6 and 24 hours. Different concentrations of aprotinin （1500,3000,6000,12000 U/ml） were then added to the third or fourth generation of cultured fibroblasts. Aprotinin was not added to the control group. MTT assay at 492 nm was used to evaluate the effect of aprrotinin on cell proliferation. Enzyme Linked Immunosorbent Assay （ELISA） at 450 nm was used to evaluate the expression of TNF-α in the cells. Results Aprotinin did not stimulate pterygium fibroblast proliferation in the experiment. However, it could inhibit the TNF-α expression of pterygium fibroblasts at different concentrations; especially at 1500 U/ml and 3000 U/ml （ P 〈 0.05 ）. The amount of suppression reached 19.36,38.46,56.15 and 13.84 pg/ml and 10.2,31.46,49.15 and 4.62 pg/ml, respectively. And the effective times for adding hydrogen peraxide were 30 min,60min,6 h and 24 h（P 〈 0.05）. Conclusion Aprotinin can inhibit the expression of TNF-α in pterygium, but not the proliferation of pterygium fibroblasts.