Vrednotenje sposobnosti adipogeneze ter stopnje staranja mišičnih in kostnih mezenhimskih matičnih celic bolnikov z osteoporozo in osteoartrozo, in vitro

Poženel, Laura (avtor), Jeras, Matjaž (mentor), Zupan, Janja (komentor)

Mezenhimske matične/ stromalne celice (MSC) izvirajo iz zarodne plasti mezoderma in predstavljajo celice, iz katerih med embrionalnim razvojem nastanejo vezivna tkiva: kostno tkivo, maščoba, hrustanec in mišice. MSC najdemo v številnih tkivih, kjer se nahajajo v tako imenovanih nišah, pri čemer so pri odraslem človeku prisotne le v majhnem številu. Njihovi glavni lastnosti sta samoobnavljanje in diferenciacija v specializirane celične linije, kar omogoča regeneracijo tkiv. So izjemno heterogena celična populacija, zato je pri njihovi izolaciji potrebno upoštevati definirane identifikacijske kriterije. MSC izolirane iz trabekularne kosti (KMSC) in skeletne mišice (MMSC) so pomemben predmet raziskav degenerativnih mišično-skeletnih bolezni, med katere spadata tudi osteoartroza in osteoporoza. Vloga kostnih in mišičnih MSC pri razvoju obeh bolezni še ni povsem znana, zato smo se v projektu, katerega del je magistrska naloga, usmerili v raziskave teh celic in vitro pri osteoartroznih in osteoporoznih bolnikih. V okviru magistrske naloge smo se osredotočili na vrednotenje sposobnosti adipogeneze in stopnje staranja kostnih in mišičnih MSC oseb z osteoartrozo in osteoporozo, in vitro. Za vrednotenje sposobnosti adipogeneze smo MSC bolnikov gojili v gojišču, ki inducira adipogenezo. Po 21 dneh smo izvedli histološko barvanje z barvilom Oil Red O ter s pomočjo programa ImageJ prešteli adipocite, ki so specifično privzeli to barvilo. Sposobnost adipogene diferenciacije smo izrazili kot odstotek adipocitov na število v kulturo naseljenih celic. Kvantifikacijo adipogeneze smo poskusili optimizirati tudi z ekstrakcijo in merjenjem absorbance barvila Oil Red O. Stopnjo staranja MSC pa smo določili s histokemijskim določanjem s staranjem povezane ß-galaktozidazne aktivnosti (SA-ß-Gal) in kvantifikacijo SA-ß-Gal-pozitivnih celic s pomočjo programa ImageJ. Stopnjo staranja smo izrazili kot odstotek SA-ß-Gal-pozitivnih celic na število gojenih celic. Dokazali smo, da je sposobnost adipogene diferenciacije KMSC in MMSC večja pri preiskovancih z osteoporozo, kot pri tistih z osteoartrozo, pri čemer pa so MMSC izkazovale večjo sposobnost adipogeneze. Menimo, da razlike v adipogeni diferenciaciji kažejo na pomembno vlogo MSC pri razvoju osteoartroze in osteoporoze. Ugotovili smo tudi, da je bila stopnja staranja kostnih in mišičnih MSC osteoartroznih bolnikov enaka, razlikovala pa se je med zgodnjimi in kasnejšimi pasažami gojenih celic.

• Jezik: Slovenski jezik
• Ključne besede: mezenhimske matične/ stromalne celice (MSC), kostne MSC (KMSC), mišične MSC (MMSC), osteoartroza, osteoporoza
• Vrsta gradiva: Magistrsko delo/naloga
• Organizacija: FFA - Fakulteta za farmacijo
• Leto izida: 2019
• PID: 20.500.12556/RUL-106513
• Datum objave v RUL: 28.02.2019

POŽENEL, Laura, 2019, Vrednotenje sposobnosti adipogeneze ter stopnje staranja mišičnih in kostnih mezenhimskih matičnih celic bolnikov z osteoporozo in osteoartrozo,  in vitro [na spletu]. Magistrsko delo. [Dostopano 21 april 2025]. Pridobljeno s: https://repozitorij.uni-lj.si/IzpisGradiva.php?lang=slv&id=106513

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: In vitro assessment of adipogenic capacity and degree of senescence of muscular and bone mesenchymal stem cells from osteoartritic and osteoporotic patients

Izvleček

Mesenchymal stem/ stromal cells (MSCs) are of mesodermal origin and represent the cells which during embryogenesis give rise to connective tissues: bone tissue, adipose tissue, cartilage and muscle tissue. MSCs are a rare population in human adults, found in numerous tissues in microenvironment called niche. Self-renewal and differentiation into specialised cell lines are two main characteristics of MSCs which enable tissue regeneration. Due to heterogenous population of these cells, a defined criteria has to be considered during MSCs isolation. MSCs isolated from trabecular bone and skeletal muscle are of great importance in research of degenerative musculoskeletal diseases, such as osteoarthritis and osteoporosis. How MSCs affect the progress of these two diseases is yet to be discovered, thus our research is focused on in vitro assessment of adipogenic capacity and degree of senescence of bone and muscular MSCs from individuals with osteoarthritis and osteoporosis. We performed histological staining of adipocytes with Oil Red O after we cultured MSCs of patients in adipogenic medium for 21 days. Further, quantification with ImageJ was made for assessment of adipogenic capacity, counting the specifically stained adipocytes. Adipogenic capacity was then determined as percentage of adipocytes on seeding cell number. Furthermore, we tried to optimize the quantification of adipogenic capacity by extracting and measuring absorbance of Oil Red O. To determine degree of senescence, we performed histochemical senescence-associated ß-galactosidase activity test (SA-ß-Gal) and quantification of SA-ß-Gal-positive cells with ImageJ. The degree of senescence was determined as percentage of SA-ß-Gal-positive cells on number of cultured cells. Our results showed that adipogenic capacity of bone and muscular MSCs is higher in osteoporotic patients, in comparison to those with osteoarthritis. Muscular MSCs had higher capacity of adipogenesis in both musculoskeletal diseases considered in our research. We believe that different capacity of adipogenesis alludes to MSCs playing an important role in progress of osteoarthritis and osteoporosis. We observed that degree of senescence is indifferent in bone and muscular MSCs in osteoartritic patients, however early and late passages of MSCs show differences in degree of senescence.

• Ključne besede: mesenchymal stem/ stromal cells (MSCs), bone MSCs, muscle MSCs, osteoarthritis, osteoporosis