Medizinische Universität Graz Austria/Österreich - Forschungsportal - Medical University of Graz
Gewählte Publikation:
Halbartschlager, F.
Effects of recombinant leptin on immortalized human chondrocyte cell lines C28/I2 and T/C-28a2 - An experimental analysis
Humanmedizin; [ Diplomarbeit ] Graz Medical University; 2017. pp.
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- Autor*innen der Med Uni Graz:
- Betreuer*innen:
- Lohberger Birgit
- Steinecker-Frohnwieser Bibiane
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- Abstract:
- Background: Osteoarthritis (OA) is a degenerative joint disease and one of the most frequent causes of musculoskeletal disability and pain around the world. Obesity is an important risk factor, not only the weight-related joint destruction, but also the highly inflammatory state in the joints of obese individuals, which leads to an imbalance of anabolic and catabolic factors in articular cartilage. Increasing evidence support the idea of leptin, an adipocytokine, being the non-mechanical link between morbid obesity, joint integrity and OA. Objectives: To investigate the effect of recombinant leptin on the cell proliferation and the expression of anabolic and catabolic components in the immortalized human chondrocyte cell lines C28/I2 and T/C-28a2. Methods: Characterization of the C28/I2 and T/C-28a2 cell lines was performed via STR- analyses. Additionally a specific vimentin-DAPI immunofluorescence imaging was performed to confirm the mesenchymal origin of the cells. The human chondrocyte cell lines were treated with recombinant leptin and their cell viability and growth behavior was examined by using a MTS- cell viability assay and a xCELLigence RD device system. Furthermore the gene expression levels of anabolic and catabolic cartilage-specific components (MMP-1, MMP-3, MMP-9, MMP-13, collagen 1A1 and collagen 2A1) were investigated by real-time PCR in order to gain information about chondrocyte responsiveness to leptin after a time period of 48 h. The effects of leptin on the expression of collagen and sGAG on protein levels were investigated by colorimetric measurements. Results: The growth behavior was recorded for 77 h and indicated that C28/I2 as well as T/C-28a2 need foetal bovine serum (FBS) essentially to grow regularly. Moreover the MTS tests showed, that the leptin treatment [0.001 µg/ml; 0.01 µg/ml; 0.1 µg/ml; 0.5 µg/ml; 1.0 µg/ml] for 24 h and 48 h has no significant negative effect on the cell viability of the cell lines. In addition the real-time PCR results showed a significant decrease of the MMP-1 and MMP-13 gene expression levels in T/C-28a2 cells when treated with 0.2 µg/ml leptin. Collagen 1A1 has a significant decreased expression in C28/I2 cells after the treatment with leptin [0.2 µg/ml]. Whereas collagen 2A1 was detected to have a decreasing tendency in both cell lines after the treatment with leptin [0.2 µg/ml and 0.5 µg/ml]. Additionally protein levels of collagen were surveyed to be slightly up regulated in the leptin + IL-1ß setup (C28/I2), whereas C28/I2 cells without IL-1ß showed a low decrease of collagen. No significant changes in the protein expression of sulfated glycosaminoglycan (sGAG) in C28/I2, but a declining leptin + IL-1ß induced tendency of sGAG were analyzed. Discussion: Our data provide interesting insights in the OA pathogenesis and the relationship between OA and obesity. Further in vitro studies are required to elucidate the complex mechanisms of leptin and to find new targets in the therapy of obesity induced OA.