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Primerjava izražanja različnih mediatorjev vnetja v mezenhimskih matičnih/stromalnih celicah pri bolnikih brez in z zgodnjimi degenerativnimi spremembami kolka
ID Mužina, Karolina (Author), ID Zupan, Janja (Mentor) More about this mentor... This link opens in a new window, ID Stražar, Klemen (Co-mentor)

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Abstract
Mezenhimske matične/stromalne celice so heterogena populacija matičnih celic, ki se nahajajo v tkivih odraslih in imajo edinstveno sposobnost multilinijske diferenciacije oz. pretvorbe znotraj mezodermalne zarodne plasti. Iz slednje se med embrionalnim razvojem razvijejo kostno tkivo, hrustanec, mišice, maščobno tkivo in druga vezivna tkiva. Mezenhimske/stromalne matične celice (MSC, angl. mesenchymal stem/stromal cells) so že nekaj časa najpogostejša tarča raziskav na področju regenerativne medicine. S poznavanjem njihove fiziološke in patofiziološke vloge pri degenerativnih boleznih kolka se kaže njihova potencialna uporaba v diagnostiki teh bolezni. Najpogostejše degenerativne bolezni mišično-skeletnega sistema so osteoporoza, osteoartoza (OA) in sarkopenija. OA je raznolika in večfaktorska bolezen sinovijskih sklepov, pri kateri sodelujejo tudi vnetni dejavniki. Osnova bolezni je napredujoča izguba strukture in popravljalne funkcije sklepa z endogenim regenerativnim procesom, ki naj bi bil pri omenjeni bolezni oslabljen. Namreč, pri mlajših zdravih posameznikih regenerativni proces po poškodbi tkiv izvedejo prav MSC. Namen magistrske naloge je bil ugotoviti razlike v izražanju genov za mediatorje vnetja v MSC, pridobljenih iz različnih tkiv kolka preiskovancev, z in brez degenerativnih sprememb. Tekom artoskopije kolka so bila preiskovancem odvzeta tri tkiva sinovije na različnih mestih kolka (S1-S3) in stebriček trabekularne kosti femurja (KO). Primarne MSC so nato namnožili v pogojih in vitro, iz njih izolirali ribonukleinsko kislino, mi pa smo nato optimizirali metodo kvantitativne verižne reakcije s polimerazo (qPCR) za merjenje izražanja genov za mediatorje vnetja. Na podlagi rezultatov optimizacije smo za nadaljnjo analizo izbrali gene TGFB1, IL6, COMP, MMP13 in VEGFA. Rezultate pridobljene s qPCR smo normalizirali glede na izbrani referenčni gen GAPDH. Rezultate smo nato primerjali med bolniki z zgodnjo OA in tistimi brez nje ter med različnimi tkivi. Preverili smo 3 hipoteze, in sicer: izražanje genov za vnetne molekule se razlikuje med MSC bolnikov z zgodnjo OA v primerjavi s tistimi brez OA, izražanje genov za vnetne molekule se razlikuje med MSC iz različnih tkiv bolnikov z zgodnjo OA in izražanje genov za vnetne molekule se razlikuje med MSC iz različnih tkiv bolnikov brez OA. Statistična analiza rezultatov ni pokazala statistično značilnih razlik v izražanju preiskovanih genov, zato smo vse 3 hipoteze zavrnili. Iz tega lahko zaključimo, da se MSC iz različnih tkiv kolka pri bolnikih z OA in pri tistih brez degenerativnih sprememb kolka ne razlikujejo v izražanju genov za vnetne molekule in da je verjetno razlog za razlike med MSC v drugih molekulah. Pomanjkljivost te študije je predvsem majhna skupina vzorcev, zato predlagamo raziskavo še na večji populaciji, da bi bila moč statističnega testa večja.

Language:Slovenian
Keywords:mezenhimske matične celice, osteoartroza, izražanje genov, qPCR, vnetni dejavniki
Work type:Master's thesis/paper
Organization:FFA - Faculty of Pharmacy
Year:2023
PID:20.500.12556/RUL-144037 This link opens in a new window
Publication date in RUL:27.01.2023
Views:381
Downloads:104
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Secondary language

Language:English
Title:The comparison of the expression of different mediators of inflammation in mesenchymal stem/stromal cells in patients without and with early degenerative hip changes
Abstract:
Mesenchymal stem/stromal cells are a heterogeneous population of stem cells that are found in adult tissues and have the unique ability of multilineage differentiation, in particular the differentiation within the mesoderm. During embryonic development connective tissues such as cartilage, muscles, fat and others develop from the mesoderm. Mesenchymal stem/stromal cells (MSCs) have been the most common research target in the field of regenerative medicine for some time. Their physiological and pathophysiological role in degenerative disorders of the hip shows their potential use in the diagnosis of these disorders. The most common degenerative disorders of the musculoskeletal system are osteoporosis, osteoarthritis (OA) and sarcopenia. OA is a diverse and multifactorial disorder of the synovial joints, where inflammatory factors are also involved. The basis of the disease is the progressive loss of the structure and repair function of the joint through the endogenous regenerative process, which is suggested to be weakened in the aforementioned disease. Namely, in younger healthy individuals, the regenerative process after tissue damage is carried out by MSCs. The aim of the master's thesis was to determine differences in the expression of genes encoding mediators of inflammation in MSCs obtained from different hip tissues of subjects with and without degenerative changes. During arthroscopy of the hip, synovium at 3 different locations of the hip (S1-S3) and a pillar of the trabecular bone of the femur (KO) were taken from the subjects. Primary MSCs were then isolated from these tissues and cultured in vitro and RNA was isolated from culture expanded cells. We then optimized a quantitative polymerase chain reaction (qPCR) method to measure the expression of genes for inflammatory mediators. Based on the optimization results, we selected TGFB1, IL6, COMP, MMP13 and VEGFA genes for further analysis. The results obtained by qPCR were normalized to reference gene GAPDH. The results were then compared between patients with early OA and those without, and between different tissues. We tested 3 hypotheses, namely: gene expression of inflammatory molecules differs between MSCs from patients with early OA compared to those without OA; gene expression of inflammatory molecules differs between MSCs from different tissues of patients with early OA and gene expression of inflammatory molecules differs between MSCs from different tissues of patients without OA. Statistical analysis of the results did not show statistically significant differences in the expression of the investigated genes, so we rejected all 3 hypotheses. Based on these results we can conclude that MSCs from different tissues of the hip and patients with OA and those without hip degenerative changes do not differ in the expression of genes for inflammatory molecules. The differences between these MSCs are probably result of other molecules. The limitation of this study is also small group of samples, so we suggest further research in larger population in order to increase the power of the statistical test.

Keywords:mesenchymal stem cells, osteoarthritis, gene expression, qPCR, inflammatory mediators

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