肿瘤坏死因子前体转换酶前肽结构域对TACE活性调节作用

The regulation of the TACE pro-domain to tumor necrosis factor-α converting enzyme activity

目的:探讨肿瘤坏死因子前体转换酶(TACE)的前肽结构域在TACE成熟过程中所起的作用,为人工干预炎症过程提供依据和手段。方法:以pIRES2-EGFP质粒为载体,利用DNA重组技术分别构造含信号肽结构域+前肽结构域、全长结构域及缺失前肽结构域的真核表达重组体,根据其碱基数目分别命名为pIRES2-EGFP/T648、pIRES2-EGFP/T2472、pIRES2-EGFP/T57-T1824;真核转染U937细胞;LPS刺激转染细胞后,用ELISA法及流式细胞技术检测TNF-α。结果:pIRES2-EGFP/T648的真核表达能显著抑制TACE的活性,减少sTNF-α分泌,抑制率达61.09%;pIRES2-EGFP/T57-T1824的真核表达对sTNF-α的分泌无影响;pIRES2-EGFP/T2472的真核表达能显著增加sTNF-α分泌。结论:前肽结构域在TACE的成熟过程中起了双重作用,TACE抑制剂的研制和开发为抗炎药物的设计和改造提供了新的依据和方法。

AIM ： To study functions of pro - domain in tumor necrosis factor - α converting enzyme （TACE） maturation and to develop an approach to interfere with inflammation processes. METI＇IODS ： The plasmid pIRES2 - EGFP was used to generate the expression plasmids constructed with the signal peptide and pro - domain, full length or without the pro - domain （ designated as plRES2 - EGFp/T648, pIRES2 - EGFP/T2472, pIRES2 - EGFP/T57 - T1824, respectively）. The plasmids were used to transfect the U937 cells. Mter the transfected cells were stimulated by LPS, the effect of expression plasmids on TNF - α secretion was detected by ELISA and flow cytometry （FCM）. RESULTS ： The plasmid plRES2 - EGFp/T648 inhibited 61.09% TNF - α secretion and mediated the accumulation of TNF - α on the surface of U937 cells. The sTNF - α secretion and the level of the mTNF - α in the plasmid plRES2 - EGFP/T57 - T1824 transfected cells had no significant deviation compared with the plRES2 - EGFP transfected cells. The sTNF - α secretion of the cells transfected by the plasmid plRES2 - EGFP/T2472 increased and the level of the mTNF - α decreased. CONCLUSION： The pro - domain has dual effects in TACE maturation and might be applied to molecular design of anti - inflammation drug.