Thesis (MSc)--University of Stellenbosch, 2007. / ENGLISH ABSTRACT: Tumour necrosis factor alpha (TNFα) is a pleiotropic cytokine and has a wide variety
of dose dependent cellular effects ranging from cell growth and differentiation, to
inducing apoptosis. It has long been implicated in muscle and non-muscle
inflammatory disorders, such as muscle wasting in chronic disease states, and
rheumatoid arthritis. However, a physiological role for TNFα in muscle regeneration
has been proposed as elevated levels of the cytokine are present when muscle
regeneration processes are initiated: TNFα is secreted by infiltrating inflammatory
cells, and by injured muscle fibres. Adult skeletal muscle contains a population of
resident stem cell-like cells called satellite cells, which become activated, proliferate
and differentiate following muscle injury to bring about repair of damaged muscle.
Much research on the effects of TNFα on satellite cell differentiation has been
conducted in recent years. It is however difficult to get a complete characterisation of
the cytokine’s action as all models used slightly differ. We aimed therefore at providing
comprehensive assessment of the effects of increasing doses of chronically
supplemented TNFα on differentiating C2C12 cells. Cells were allowed to differentiate
with or without TNFα supplementation for 7 days. Differentiation was induced at day
0. The effect on differentiation was assessed at days 1, 3, 5, and 7 by western blot
analysis, and supplementary immunohistochemical analysis at days 1, 4, and 7 of
markers of differentiation - muscle regulatory factors: MyoD and myogenin, markers of
the cell cycle p21, PCNA, and the integral signalling molecule, p38MAPK. TNFα
supplementation at day 1 tended to positively regulate early markers of differentiation.
With continued supplementation however, markers of differentiation decreased dose
dependently in treated cultures as the initial effect appeared to be reversed: A trend
towards a dose dependent decrease in MyoD, myogenin and p21 protein existed in treated cultures at days 3, 5, and 7. These findings were significant at day 5 (p21,
p<0.05), and day 7 (myogenin, p<0.05). A significant dose dependent decrease in p38
phosphorylation was evident at day 3 (p<0.05), while phospho-p38 was dose
dependently increased at day 7 (p<0.05). Taken together, these data show that TNFα
supplementation for 24 hours following the induction of differentiation in vitro, tends
to increase levels of early markers of differentiation, and with continued TNFα
supplementation decrease markers of differentiation in a dose dependent fashion. This
study provides a comprehensive characterisation of the dose and time dependent effects
of TNFα on satellite cell differentiaton in vitro. The model system used in the current
study, allows us to make conclusions on more chronic disease states. / AFRIKAANSE OPSOMMING: Tumor nekrose faktor alfa (TNFα) is ‘n pleiotropiese sitokien wat ‘n wye
verskeidenheid, dosis afhanklike, sellulêre effekte te weeg bring. Hierdie sellulêre
effekte sluit sel groei en differensiasie tot sel dood in. TNFα is by beide spier en niespier
inflammatoriese stoornisse soos spier tering in kroniese siektetoestande, en
rumatiese artritis betrek. ‘n Fisiologiese rol vir TNFα is egter voorgestel aangesien
verhoogde vlakke van die sitokien tydens inisiasie van spier herstel meganismes
teenwoordig is: TNFα word deur infiltrerende inflammatoriese selle, asook deur
beseerde spier vesels afgeskei. Volwasse skeletspier bevat ‘n populasie stamselagtige
selle, sogenoemde satelliet selle. Laasgenoemde word geaktiveer, prolifereer en
differensieër volgende spierbesering, om sodoende herstel van beskadigde spier te
weeg te bring. Baie navorsing op die effekte van TNFα op satelliet sel differensiasie is
onlangs uitgevoer. Dit is egter aansienlik moeilik om volgens hierdie navorsing‘n
algehele beeld van TNFα se aksies te vorm aangesien alle modelle wat gebruik word
verskil. Ons doel was daarom om ‘n omvangryke assessering van toenemende
konsentrasies kronies gesupplementeerde TNFα op differensieërende C2C12 selle op ‘n
enkele model uit te voer. Selle was vir 7 dae met of sonder TNFα supplementasie
gedifferentieër. Differensiasie was by Dag 0 geïnduseer. TNFα se effek op
differensiasie is op dae 1, 3, 5, en 7 deur middel van western blot analise geassesseer.
Aanvullende immunohistochemiese bepalings op dae 1, 4, en 7 is verder deurgevoer.
Merkers vir differensiasie het die spier regulatoriese faktore MyoD en miogenien, sel
siklus merkers p21 en PCNA, asook die integrale sein transduksie molekule p38MAPK
ingesluit. TNFα supplementasie by dag 1 het geneig om vroeë merkers van
differensiasie positief te reguleer. Met voortdurende supplementasie is die vroeë
positiewe effekte (op ‘n dosis afhanklike manier) egter omgekeer: ‘n neiging teenoor (‘n dosis afhanklike) vermindering in MyoD, miogenien en p21 proteïen het in
behandelde kulture op dae 3, 5, en 7 bestaan. Hierdie bevindinge was beduidend by dag
5 (p21, p<0.05), en dag 7 (miogenien, p<0.05). A beduidende dosis afhanklike afname
in p38 fosforilasie was duidelik by dag 3 (p<0.05), terwyl fosfo-p38 by dag 7 verhoog
het met verhoogde konsentrasie TNFα (p<0.05). Bogenoemde saamgevat, dui aan dat
TNFα supplementasie 24h volgende die induksie van differensiasie in vitro, verhoogde
vlakke van vroeë differnsiasie merkers te weeg bring. Met voortdurende TNFα
supplementasie, word differensiasie merkers egter met toenemende dosis verminder.
Hierdie studie voorsien ‘n omvattende karakterisering van die dosis- en tyd afhanklike
effekte van TNFα op satelliet sel differesiasie in vitro. Die model sisteem in hierdie
studie gebruik, maak afleidings oor meer kroniese siektetoestande moontlik.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/19596 |
| Date | 03 1900 |
| Creators | Fouche, Celeste |
| Contributors | Smith, Robert M., Niesler, Carola U., Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | Unknown |
| Type | Thesis |
| Format | 102 leaves : ill. |
| Rights | Stellenbosch University |
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