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首页> 外文期刊>Osteoarthritis and cartilage >Effect of adenovirus-mediated overexpression of bovine ADAMTS-4 and human ADAMTS-5 in primary bovine articular chondrocyte pellet culture system.
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Effect of adenovirus-mediated overexpression of bovine ADAMTS-4 and human ADAMTS-5 in primary bovine articular chondrocyte pellet culture system.

机译：腺病毒介导的牛ADAMTS-4和人ADAMTS-5在原代牛关节软骨细胞沉淀培养系统中的过表达。

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INTRODUCTION: Articular cartilage matrix synthesis and degradation are dynamic processes that must be balanced for proper maintenance of the tissue. In osteoarthritis (OA), this balance is skewed toward degradation and ultimate loss of matrix. The transcriptional and/or activity levels of hundreds of genes are dysregulated in chondrocytes from osteoarthritic cartilage, and a subset of these genes may represent pivotal factors that could be modulated if their specific role in the disease process could be identified. OBJECTIVE: To investigate the role of ADAMTS-4 and ADAMTS-5 in cartilage matrix degradation by developing a chondrocyte pellet culture assay in combination with adenoviral gene expression, and to demonstrate the utility of this assay by assessing the specific functional contribution of these genes to cartilage matrix metabolism. METHODS: A full-length cDNA for bovine ADAMTS-4 (bADAMTS-4) was isolated, and used to evaluate the expression, regulation, and activity of this gene in bovine cartilage. Adenoviruses expressing bADAMTS-4, human ADAMTS-5 (hADAMTS-5) or human bone morphogenetic protein 2 (BMP-2) were used to infect primary chondrocytes, and their effect on extracellular matrix metabolism was assessed by monitoring the accumulation and release of glycosaminoglycans (GAG) in three-dimensional chondrocyte pellet cultures. RESULTS: Analysis of bADAMTS-4 transcriptional regulation in chondrocytes revealed that interleukin-1alpha (IL-1alpha) was the most potent inducer of bADAMTS-4 mRNA and subsequent aggrecan degradation in cartilage explant cultures of those cytokines tested. bADAMTS-4 mRNA induction by IL-1alpha was greater in nasal cartilage than in articular cartilage. Chondrocytes infected with adenovirus expressing either bADAMTS-4 or hADAMTS-5 genes showed increased aggrecan degradation in newly synthesized matrix by pellet cultures while chondrocytes overexpressing BMP-2 showed increased aggrecan synthesis. CONCLUSION: Adenoviral delivery of genes to primary bovine chondrocytes, followed by culture in three-dimensional pellet format and evaluation of extracellular matrix protein metabolism, is a useful functional assay for assessing the role of genes on cartilage matrix synthesis and degradation.
机译：简介：关节软骨基质的合成和降解是动态过程，必须适当平衡才能正确维护组织。在骨关节炎（OA）中，这种平衡偏向于降解和基质的最终损失。数百种基因的转录和/或活性水平在骨关节炎软骨细胞中失调，如果能够确定它们在疾病过程中的特定作用，这些基因的一个子集可能代表着可以调节的关键因素。目的：通过开发软骨细胞沉淀培养法并结合腺病毒基因表达来研究ADAMTS-4和ADAMTS-5在软骨基质降解中的作用，并通过评估这些基因对腺病毒的特定功能贡献来证明该方法的实用性软骨基质代谢。方法：分离出牛ADAMTS-4的全长cDNA（bADAMTS-4），并用于评估该基因在牛软骨中的表达，调控和活性。表达bADAMTS-4，人ADAMTS-5（hADAMTS-5）或人骨形态发生蛋白2（BMP-2）的腺病毒用于感染原代软骨细胞，并通过监测糖胺聚糖的积累和释放来评估其对细胞外基质代谢的影响。 （GAG）在三维软骨细胞沉淀培养物中。结果：对软骨细胞中bADAMTS-4转录调控的分析表明，白介素-1α（IL-1alpha）是bADAMTS-4 mRNA的最强诱导剂，随后在这些受试细胞因子的软骨外植体培养物中聚集蛋白聚糖降解。 IL-1α诱导的bADAMTS-4 mRNA在鼻软骨中的表达高于在关节软骨中的表达。感染了表达bADAMTS-4或hADAMTS-5基因的腺病毒的软骨细胞通过沉淀培养在新合成的基质中聚集蛋白聚糖降解增加，而过表达BMP-2的软骨细胞表达聚集蛋白聚糖则增加。结论：腺病毒向原代牛软骨细胞传递基因，然后以三维沉淀形式培养并评估细胞外基质蛋白代谢，是评估基因在软骨基质合成和降解中作用的有用的功能检测方法。

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