三类TACE抑制剂的作用机理探讨

The study on different acting mechanisms of three types of TACE inhibitors in converting of pro-TNFα into sTNFα

目的 :以脂多糖 (LPS)刺激的HL 6 0细胞系为天然模型 ,筛选肿瘤坏死因子α转换酶的抑制剂 ,为人工干预炎症过程提供依据和手段。方法 :分时相刺激HL 6 0细胞后 ,用分子生物学和细胞学技术在蛋白质和mRNA水平上研究TACE表达和活性的改变。结果 :中药热毒清 (RDQ)能明显降低LPS诱导的TACEmRNA增加 (与对照组比 ,P <0 .0 1) ;针对TACEmRNA的反义寡核苷酸 (anti ODN)在翻译水平上抑制TACE表达 ,抑制率达 78.9% ;化学合成的针对TACE催化域的小分子底物模拟肽可抑制LPS诱导的TACE表达增加 ,从而抑制sTNFα分泌增加 ,削弱LPS诱发的炎性病理反应强度。结论 :3种不同类型的抑制 (RDQ、anti ODN、底物模拟肽 )在不同层次上调节TACE的表达 ,最终均抑制了sTNFα的分泌 ,为控制炎症提供了明确的治疗靶标。

Objective:To study the effects of TNFα converting enzyme(TACE)inhibitors on TNFα secretion and develop an approach to interfere inflammation processes.Methods:(1)Stimulate the HL-60 cell lines in vitro with LPS for different time to establish the cellular model of inflammation and simultaneously induce in vivo inflammatoin animal model by LPS.(2)Check the cytotoxic effects of TNFα secretion using MTT colorimetric method for cell proliferaton.(3)Detect the level of expression of TACE carrying out RT-PCR,FCM techniques and immuno-histochemical dying technique.Results:(1)PDQ had the inhibitive effect on TNFαmRNA expression induced by LPS stimulation( P<0.01, compared with the control).(2)The anti-oligodeoxynucleotides (anti-ODN)can inhibit 78.9% TNFα secretion.(3)The hydroxamic acids derived mimic peptides of TACE substrates showed potency against in vivo and in vitro releasing of pro-TNFα.Conclusion:The experiments prove all the three types of TACE inhibitors can adjust the expression of TACE in different levels and inhibit sTNFα secretion in the end,which indicates TACE is a novel target for inflammation therapy.