Molecular diagnostic approach to determine the degree of photoaging of the skin

Thesis (MScMedSc)--Stellenbosch University, 2015.ENGLISH ABSTRACT: Context: Excessive exposure to ultraviolet radiation (UV) results in the risk of acquiring long-term harmful effects such as photoaging, which is characterised by deep wrinkles, roughness, dyspigmentation and an increased loss in elasticity. As a result, the detection of photoaging at an early stage is crucial to improving morbidity, whilst preventing the advancement of skin cancer. Aim: The aim of the study was to develop and to validate a diagnostic real-time PCR method in order to establish the gene expression profiles of potential biomarkers in the skin so as to quantify the degree of photoaging: this was conducted by retrieving total RNA from cells adherent to tape strips from sun exposed and non-exposed skin areas. Materials and methods: Twenty healthy volunteers consisting of seven males and thirteen females aged 25 to 67 years were included in this study. Tape stripping was performed using pre-cut D-Squame® 22 mm adhesive discs. Samples were collected on the right medial thigh area 20 cm above the patella and 2 cm below the lateral canthus of the right eye. Total RNA was extracted and relative standard curve method of gene expression was performed. TGF-β, MMP 9, TNF-α and IL-6 mRNA transcripts were selected as representative cytokines to determine the relative fold-change in sun exposed and non-exposed areas of the skin so as to determine extent of photoaging. Results: Repeatability and reproducibility was determined by the coefficient of variation (CV) was within an acceptable range. Thirty five percent (n=7) samples displayed down-regulatory effects for TGF-β. Down regulation of MMP 9 was observed within 30% (n=6) of samples, while 15% (n=3) showed marked up regulation. Only two samples showed measurable levels of TNF-α in the assay, of which one showed significant up regulation. Furthermore, we were unable to detect any IL-6 expression in any of the samples prepared. Conclusion: we have shown that epidermal cytokines can be retrieved from tape stripped samples and can be quantified via real-time PCR. However, the choices of cytokine biomarkers reveal that they are as important as the concentration of starting material. In this study cytokines such as IL-6 is not as informative in determining the extent of photoaging without high doses of ultraviolet radiation before sample collection as opposed to the other explored cytokines.AFRIKAANSE OPSOMMING: Konteks: Oormatige blootstelling aan ultraviolet (UV) bestraling kan tot ‘n risiko van skadelike en lantermynse nagevolge lei wat gekenmerk word deur foto-veroudering. Dit sluit in diep plooie, growwe vel en ‘n toenemende verlies in elastisiteit. Die ontdekking van foto-veroudering op ‘n vroeë stadium is van kardinale belang vir die verbetering van morbiditeit en die voorkoming van velkanker bevordering. Doelstelling: Die doel van hierdie studie was om ‘n diagnostiese polimerase kettings reaksie (PKR) metode te ontwikkel om geen uitdrukkings profiele van potensiële bio-merkers te vestig in die vel, om so die graad van foto-veroudering in areas van vel wat blootgestel word aan die son en beskermde van die son te bepaal deur totale RNS te versamel van kleeflintskyfies. Materiale en metodes: Twintig gesonde vrywilligers (sewe mans en dertien vroue), tussen die ouderdom van 25 en 67 jaar, was ingesluit in hiedie studie. Vel monsters was versamel deur gebruik te maak van Dsquame® 22 mm kleeflintskyfies 20 cm bokant die patella van die regterkanste mediale heup en 2 cm onder die regter oog. Totale RNS was geisoleer en die relatiewe vlak van geen uitdrukking was bepaal deur gebruik te maak van die kurwe model. Die boodskapper ribonukleiosier transkripsies van die sitokiene TGF- β, MMP 9, TNF-α en IL-6 was gekies as verteenwoordigers van foto-veroudering om die relatiewe verandering van foto-veroudering in die vel te bepaal. Resultate: Validering metodes was aanvaarbaar. ‘n Afwaarts reguleringseffek in TGF-β en MMP 9 merker uitdrukking is gevind in vyf en dertig persent (n=7) en dertig persent (n=6) van monsters, onderskuidelik. In vyftien persent (n=3) van monsters is ‘n opwaarts reguleringseffek in die laasgenoemde gevind. Slegs twee monsters het meetbare vlakke van TNF-α getoon in die eksperiment, waarvan slegs een ‘n noemenswaardige opwaartse regulering getoon het. IL-6 uitdrukking is nie gevind in enige van die monsters. Gevolgtrekkings: Hierdie studie het bepaal dat sitokiene van die vel geisoleer van kleeflint monsters en gekwantifiseer deer relatiewe PKR uitdrukking bepaal kan word. Die keuse van bio-merkers is egter net so belangrik as konsentrasie bepaling van die monsters. Die IL-6 sitokien, in vergelyking met ander, is slegs informaliet tydens hoë ultraviolet bestraling aan die vel blootgestel is