Satellite cells are quiescent skeletal muscle specific stem cells that are activated in response to injury to aid in muscle repair and regeneration. The interaction of hepatocyte growth factor (HGF) with these cells is crucial for their activation. However, to date, research on the effect of HGF on skeletal muscle satellite cells has yielded conflicting data. Clarity is therefore required as to its effect on downstream myogenic processes. Furthermore, mouse and rat cell lines and primary culture have been widely used for in vitro studies to investigate the effect of HGF on skeletal muscle physiology and disease; very few studies have been carried out in primary cultured human skeletal myoblasts. As a result, we aimed to investigate and compare the effect of HGF (2, 10 and 50 ng/ml) on mouse C2C12 myoblast versus primary culture human skeletal myoblast (HSkM) proliferation, migration and differentiation. Proliferation was assessed via both cell counts and crystal violet assay, while migration was investigated using the scratch assay. Differentiation was determined via analysis of expression patterns of transcription factors implicated in myogenic commitment (i.e. Pax7, MyoD) as well expression of the structural protein Myosin Heavy Chain (MyHC). We demonstrate a dose-dependent effect of HGF on myoblast proliferation whereby an increase in proliferation was detected in response to 2 ng/ml HGF, whilst 10 ng/ml HGF resulted in a reduction in proliferation capacity of both C2C12 and HSkM myoblasts. Interestingly, the reduction in proliferation in response to 10 ng/ml HGF was accompanied by a down-regulation in Pax7 expression during differentiation of both mouse and human myoblasts. HGF also affected myoblast migration and differentiation in a dose-dependent manner that was inversely proportional to proliferation. HGF (10 ng/ml) stimulated an increase in myogenic commitment and terminal differentiation of C2C12 and HSkM myoblasts as reflected by the increased percentage MyoD positive cells, improved fusion and greater MyHC expression. C2C12 myoblast migration was also stimulated at this HGF concentration, but reduced in response to the lower HGF (2 ng/ml) dose. The decrease in proliferation following incubation with 10 ng/ml HGF, allows cells to exit proliferation into either a mode of migration or differentiation. Our data confirms the importance of HGF during myogenesis and highlights the sensitivity of satellite cells to changing HGF concentration. This has implications in the regulation of skeletal muscle wound repair. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2013.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:ukzn/oai:http://researchspace.ukzn.ac.za:10413/10782 |
| Date | 29 May 2014 |
| Creators | Kahamba, Trish R. |
| Contributors | Niesler, Carola U. |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
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