Mutation of the Col2a1 Gene (G1170S) and Centrifugation Alter the Murine Phenotype and Cartilage Matrix Homeostasis

• Main Authors: Ruei-Cheng Yang, 楊瑞成
• Other Authors: Cheng-Kung Cheng
• Format: Others
• Language: en_US
• Published: 2013
• Online Access: http://ndltd.ncl.edu.tw/handle/25990154717973226596

博士 === 國立陽明大學 === 醫學工程研究所 === 102 === Genomic studies have revealed that there is a significant association between a point mutation of the human Col2A1 gene (G1170S) and several hip disorders, such as osteonecrosis of the femoral head, Legg-Calvé-Perthes disease and precocious osteoarthritis of the hip. Cartilage metabolism is also modulated by mechanical stimulation. The aim of our two studies was to explore the effects of genetic and mechanical factors on murine phenotype and cartilage matrix homeostasis. In the first study, wild-type mice and transgenic mice were used as the control and study groups respectively. The body weight, radiographic analysis and histological analysis of the mice were carried out to describe the differences between the wild-type mice and the transgenic mice at different ages. In the second study, two different types of murine chondrocytes (wild-type and a Col2a1 gene knock-in) were used. Short term steady centrifugation was selected as the mechanical model. Four groups, consisting of unloaded wild-type cells, loaded wild-type cells, unloaded knock-in cells and loaded knock-in cells, were used for the analysis. The study then measured cellular proliferation, assayed the secreted products of the chondrocytes, including total glycosaminoglycan (GAG) and nitric oxide (NO) and examined the gene expression levels of TNF-α, IL-1ß, IL-6, COX-2, iNOS, MMP-3, MMP-9, MMP-13, ADAMTS-4, ADAMTS-5, TIMP-1, aggrecan and Col2a1. The results of these assays were compared across the four groups. In the first study, the transgenic mice had a lower mean body weight, a deformed skeletal structure and abnormal cartilage histomorphology. In the second study, the genetic impact of the mutated Col2a1 genetic on chondrocytes (unloaded knock-in cells vs. unloaded wild-type cells) involved a significant fold change increase in TNF-α, MMP-3 and ADAMTS-4 gene expression in parallel with a significant fold change decrease in IL-1β, COX-2 and Col2a1 gene expression. Mechanical stimulation of wild-type chondrocytes (loaded vs. unloaded cells) showed a significant fold change decrease in IL-1ß and iNOS gene expression. When the combined impact of the Col2a1 mutation and mechanical stimulation on chondrocytes was assessed (loaded knock-in cells vs. unloaded wild-type cells), it was found that there was significantly increased NO secretion on day 4 of culture together with a significant fold change increase in TNF-α, IL-6, MMP-3 and ADAMTS-4 gene expression linked to a significant fold change decrease in IL-1ß and Col2a1 gene expression. In conclusion, the mutant Col2a1 gene (G1170S) in mice clearly alters the transgenic murine phenotype. When genetic and mechanical factors are combined, this makes murine cartilage matrix more vulnerable than the genetic change alone.