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背景与目的:以肿瘤坏死因子(Tumournecrosisfactor-α,TNF-α)前体向分泌型TNF-α转化过程为目标,探讨HL-60细胞中金属蛋白酶(Metalloproteinases,MPs)家族的基质金属蛋白酶(Matrixmetalloproteinases,MMPs)和解整合素金属蛋白酶(Adisintegrinandmetalloproteases,ADAM)两亚类成分参与炎症因子分泌的机理以及中药热毒清(Reduqing,RDQ)抗炎保护功效的分子机制。材料与方法:采用细胞毒性-MTT检测法、原位杂交、PAG-底物(明胶)电泳等方法分别检测HL-60、U937和小鼠腹腔巨噬细胞在大肠杆菌内毒素(LPS)及RDQ的作用下,MMPs、ADAM17、TNF-α基因转录水平和酶活性的变化。结果:①LPS刺激后,不同类型细胞的MMPs的表达量及电泳酶谱随刺激时间的延长变弱,而HL-60细胞培养上清的酶谱变化正好相反;②在HL-60细胞中与MMPs相比,ADAM17在前体TNF-α(pro-TNF-α)向分泌型TNF-α(s-TNF-α)转换中起着重要作用;③RDQ在细胞杀伤效应上、在ADAM17mRNA表达水平上均能明显抑制LPS所诱导的s-TNF-α表达和分泌的增高作用(P<0.01)。结论:①在LPS刺激的TNF-α分泌中,MPs家族的ADAM17起主要作用,而针对解整合素结构域(Disintegrin)的探针对排除MMPs的干扰,真实反映ADAM17mRNA表达水平更具特异性;②纯中药注射液RDQ的抗炎、解毒作用的靶标之一是拮抗LPS诱导的TACEmRNA活性增加。ADAM17成为炎症机制和治疗研究的新靶标。
BACKGROUND & AIM: To investigate the transformation of Tumour necrosis factor-α (TNF-α) precursors to secretory TNF-α and to investigate the role of matrix metalloproteinases in the metalloproteinases (MPs) family of HL-60 cells. , MMPs) and Adisintegrin and metalloproteases (ADAM) are two sub-components involved in the mechanism of inflammatory cytokine secretion and the molecular mechanisms of the anti-inflammatory protective efficacy of the traditional Chinese medicine Reduqing (RDQ). MATERIAL AND METHODS: To detect HL-60, U937 and mouse peritoneal macrophages in E. coli endotoxin (LPS) and RDQ using cytotoxicity-MTT assay, in situ hybridization, PAG-substrate (gelatin) electrophoresis, etc. Under the effect of MMPs, ADAM17, TNF-α gene transcription level and enzyme activity changes. RESULTS: 1 After LPS stimulation, the expression levels of MMPs and the electrophoretic patterns of different types of cells weakened with the prolongation of stimulation time, but the zymogram changes of the culture supernatant of HL-60 cells were exactly the opposite; 2 In HL-60 cells and MMPs Compared with ADAM17, ADAM17 plays an important role in the transition from pro-TNF-α to secretory TNF-α (s-TNF-α); 3RDQ has both cell killing effect and ADAM17 mRNA expression level. It can significantly inhibit the increase of s-TNF-α expression and secretion induced by LPS (P<0.01). Conclusions: 1 In TNF-α secretion stimulated by LPS, ADAM17 of MPs family plays a major role, and the interference of integrin-dissolved probes on MMPs excluding MMPs reflects the specificity of ADAM17 mRNA expression. 2 One of the targets of anti-inflammation and detoxification of pure Chinese medicine injection RDQ is to antagonize LPS-induced increase in TACE mRNA activity. ADAM17 has become a new target for inflammatory mechanisms and therapeutic research.