| Summary: | 碩士 === 國立陽明大學 === 復健科技輔具研究所 === 95 === Introduction: Point mutation of COL(collagen)2A1 gene might alter the quantity and quality of type 2 collagens, thereafter changed the microenvironment surrounding the chondrocytes. The purpose of the study is to demonstrate the influence of point mutation in Col2a1 gene on transgenic murine chondrocytes and evaluate functional change of the chondrocytes in balance between biosynthesis and degradation of extracellular matrix (ECM).
Materials and Methods: The transgene of G (Guanine) > A (Adenine) base transition responsible for Gly (Glysine) 1102Ser (Serine) codon change was constructed to breed transgenic mice. The transgenic mice were marked as the study group and the wild-typed mice were marked as the control group respectively. The 4~6 days old mice were sacrificed and cartilage lump of their knees were harvested as source of primary chondrocytes culture. Spectrophotometry included 3- (4,5- dimethyl-thiazol- 2-yl)- 2,5-diphenyltetrazolium bromide (MTT) assay and Griess reaction for nitric oxide (NO). The mRNA expression of the transgene and the normal Col2a1 gene was calibrated with TaqMan assay. The mRNA expression of tumor necrosis factor (TNF), Interleukine-1 (IL-1), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), matrix metalloproteinase (MMPs), tissue inhibitor of metalloproteinase (TIMPs), and a disintegrin and metalloproteinase with
thrombospondin motifs (ADAMTS) was detected with real time reverse transcriptase-polymerase chain reaction (RT-PCR). Pared Student t-test was use as the statistical method. P < 0.05 was designated as significant difference.
Results: MTT assay demonstrated that the optic density (OD) value in the control group is significantly higher than that in the study group at day 4, day7, and day14. NO assay demonstrated that the OD value in the study group was significantly higher than that in the control group at day1 and day14 respectively. The TaqMan assay revealed that the mRNA expression of the normal Col2a1 gene in the study group was 0.41 times that of the normal Col2a1 gene in the control group and the transgene in the study group was 0.96 times that of the normal Col2a1 gene in the control group. The real time RT-PCR showed that mRNA expression of IL-1β in the study group was 1.72 times that in the control group and mRNA expression of TNF-α in the study group was 1.47 times that in the control group.
Discussion: Point mutation of Col2a1 gene in the transgenic mice only enhances local inflammation via chondrocyte-originated endogenous cytokine pathway. There are no significantly changed activities of the enzymes in charge of balance between biosynthesis and degradation of ECM surrounding the chondrocytes.
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