Vpliv različnih načinov dodajanja teriparatida na izražanje osteogenih genov v mezenhimskih matičnih celicah

Štaman, Melanija (author), Ostanek, Barbara (mentor), Vrščaj, Lucija Ana (co-mentor)

Osteoporoza je bolezen, pri kateri gre za zmanjšanje kostne gostote, kar vodi do povečanega tveganja za zlome kosti, predvsem za zlome v predelu kolka in vretenc. Za zdravljenje osteoporoze najpogosteje uporabljamo antiresorpcijska zdravila in redkeje osteoanabolna zdravila. Med osteoanabolne zdravilne učinkovine spada tudi teriparatid, ki je analog parathormona (PTH) in je sestavljen iz prvih 34 N-terminalnih aminokislin (PTH 1-34). Način odmerjanja teriparatida določa, kakšen učinek ima na kosti. Če teriparatid odmerjamo enkrat dnevno oziroma intermitentno, ima anabolen učinek, kar pomeni, da stimulira izgradnjo kosti, če pa teriparatid odmerjamo kontinuirano pa ima katabolen učinek in stimulira razgradnjo kosti. Teriparatid deluje na mezenhimske matične celice (MSC), matične celice iz maščobnega tkiva (ADSC), na predhodnike osteoblastov ter na zrele osteoblaste, na osteocite ter na limfocite T. MSC so multipotentne celice, ki lahko diferencirajo v različne celice, v katero celično vrsto se MSC usmeri pa je odvisno od transkripcijskih dejavnikov in citokinov, prav tako pa so vključene tudi številne signalne poti. Nekatere molekule, ki so pomembne pri diferenciaciji MSC v osteoblastne celice uporabimo kot označevalce diferenciacije MSC v osteoblastno linijo. Med najpomembnejšimi označevalci osteoblastogeneze so RUNX2, ALP, COL1A1, OCN in OPN. Namen naloge je bil ugotoviti, kateri način tretiranja MSC in vitro najbolj spodbuja osteogeno diferenciacijo, ki je značilna za intermitentno dajanje teriparatida in vivo. Primerjali smo 6 različnih načinov tretiranj v šestih različnih časovnih točkah. Z metodo kvantitativne verižne reakcije s polimerazo v realnem času (qPCR) smo določali izražanje genov ALP, RUNX2, COL1A1, BMP2, OCN, PTH1R in referenčni gen GADPH. Pri genu ALP smo izmerili najvišje izražanje gena pri kontinuiranem tretiranju celic (TPTD 24/7). Pri genu RUNX2 ni večjih razlik med posameznimi načini tretiranja, enako velja tudi za gen COL1A1. Pri genu OCN smo opazili znižano izražanje glede na kontrolni vzorec, kar je bilo pričakovano. Pri genu BMP2 je 14. dan opazno zvišanje izražanja pri celicah, ki sm jih tretirali celoten čas gojenja (TPTD 24/7). Največjo dinamiko smo opazili pri genu PTH1R, največje zvišanje izražanja gena smo izmerili 21. dan pri celicah, ki smo jih gojili v osteogenem mediju brez dodajanja teriparatida. S pomočjo barvanja z alizarinom, ki smo ga opravili po 21 dneh tretiranja, smo potrdili, da je prišlo do diferenciacije MSC v osteoblaste. Do najintenzivnejšega obarvanja kalcijevih depozitov je prišlo pri celicah, ki smo jih tretirali intermitentno (TPTD 6 h) in pri kontinuiranem tretiranju (TPTD 24/7), kar kaže na to, da je v teh dveh primerih bila največja uspešnost osteogene diferenciacije MSC. Glede na dobljene rezultate izražanja genov ne moremo dati dokončnega zaključka, kateri način tretiranja s teriparatidom najbolj spodbuja osteogeno diferenciacijo, lahko pa rečemo, da so največje razlike opazne pri intermitentnem tretiranju s teriparatidom 6 ur (TPTD 6 ur) in pri kontinuiranem tretiranju 24/7 (TPTD 24/7). Omenjeni tretiranji bi bilo potrebno bolj natančno ovrednostiti z nadaljnjimi poskusi.

• Language: Slovenian
• Keywords: teriparatid, mezenhimske matične celice, osteoporoza, qPCR, osteoblastna diferenciacija
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2024
• PID: 20.500.12556/RUL-155559
• Publication date in RUL: 06.04.2024

Secondary language

• Language: English
• Title: Effects of different teriparatide treatments on osteogenic differentiation of mesenchymal stem cells

Abstract

Osteoporosis is a disease that develops when bone mass decreases, which leads to increased risk of bone fractures, mainly hip and spine fractures. For treatment of osteoporosis there are two options, antiresorptive or osteoanabolic therapy. Teriparatide is an anabolic drug. It is the analogue of parathyroid hormone (PTH) and it consists of first 34 N-terminal amino acids (PTH 1-34). Teriparatide has different effect on bone, depending on its administration method. Intermittent administration of teriparatide has anabolic effect on bones, and on the other hand continuous exposure to teriparatide has catabolic effect on bones. Teriparatide affects mesenchymal stem cells (MSC), adipocyte-derives stem cells (ADSC), osteoblast precursors, mature osteoblasts, and T cells. MSCs are multipotent cells that can differentiate into a variety of cell types, depending on transcriptional factors, cytokines, and signaling pathways involved. Some of those are used as markers of MSC differentiation to osteoblasts, including RUNX2, ALP, COL1A1, OCN and OPN. The aim of the study was to determine which mode of in vitro administration of teriparatide best stimulates osteogenic differentiation, which is characteristic for intermittent administration of teriparatide in vivo. We compared 6 different modes of administration in 6 different time points. We used quantitative real-time polymerase chain reaction (qPCR) to determine gene expressions of ALP, RUNX2, COL1A1, BMP2, OCN, PTH1R and reference gene GADPH. We measured the highest ALP gene expression when cells were treated continuously (TPTD 24/7). There is no differences between modes of teriparatide treatment in RUNX2 and COL1A1 gene expressions. As expected, we measured lower OCN gene expression compared to control sample. There is the highest BMP2 gene expression on 14th day, when the MSC were treated continuously (TPTD 24/7). PTH1R gene has the bigest dinamic compared to other genes. We measured the highest gene expression on 21th day, when the MSC were incubated in osteogenic medium without teriparatide. Given the results from alizarin staining, we concluded that MSC differentiated into osteoblastic cells. The most intense staining of calcium deposits occurred in cells that were treated intermittently (TPTD 6 h) and in continuously (TPTD 24/7), which indicates that these two modes of teriparatide treatment showed the greatest success of osteogenic differentiation of MSC. Depending on our results, it is not possible to determine the best mode of administration of the drug. What we can say is that the biggest differences were observed in intermittent treatment with teriparatide fo 6 hours (TPTD 6 h) and in continuous treatment 24/7 (TPTD 24/7). It is necessary to carry out aditional experiments to determine which mode of administration of teriparatide is better.

• Keywords: teriparatide, mesenchymal stem cells, osteoporosis, qPCR, osteoblast differentiation