izvor podataka: poirot

Naziv

Utjecaj koštanog morfogenetskog proteina 3 (BMP3) na regeneraciju kosti

Effect of bone morphogenetic protein 3 (BMP3) on bone regeneration

Opis projekta

Osnova pokusa će biti miševi kojima je izbačen gen za Bmp3 (KO), soja 57BL/6Ntac u kojima je u 1. egzon Bmp3 gena u okviru (in frame) ubačen konstrukt koji sadrži gen dojavljivač za β-galaktozidazu. Na taj način je dokinuto prepisivanje Bmp3 gena u protein i miševi nemaju funkcionalni Bmp3 protein. Kao kontrolni miševi služit će miševi soja 57BL/6Ntac divljeg tipa (WT). Kultura stanica U kulturi stanica će se na staničnoj liniji MC3T3 mišjih pre-osteoblasta testirati aktivnost komercijalno dostupnih BMP3 proteina i protutijela koji će se kasnije koristiti u pokusima proliferacije i diferencijacije navedenih stanica. U primarnoj kulturi matičnih stanica koštane srži izdvojene iz 57BL/6Ntac (KO i WT) miševa će se koštana srž ispuhati pomoću sterilnog staničnog medija i štrcaljke iz dijafiza dugih kostiju i nasaditi će se na pločice kako bi se proučila diferencijacija i proliferacija osteoblastičnih i osteoklastičnih stanica i kako izostanak BMP3 proteina utječe na te procese. Životinjski modeli koštanog stvaranja i regeneracije 1. Ektopično stvaranje kosti. Korištenje pune krvi kao nosača za BMP6 pokazalo se uspješnim u stvaranju ektopične kosti. U pokusima će se proučiti utjecaj BMP3 proteina na proces de novo stvaranja kosti pomoću ektopičnog eseja. Rekombinantni ljudski BMP-6 u nosaču od autologne krvi će se koristiti za provjeru osteogenog odgovora BMP3-/- miševa i miševa divljeg tipa (WT). Dodavanjem egzogenog BMP3 proteina ili BMP3 protutijela u ugrušak sa BMP6 omogućit će dodatni uvid u mehanizam djelovanja BMP3 na stvaranje kosti. Grupe životinja će biti uspostavljene prema kombinacijama BMP3 proteina, BMP3 protutijela i BMP6 proteina. Vrijeme inkubacije ugruška će biti 14 dana nakon čega će završiti pokus. Prilikom završetka pokusa miševima će biti izvađena krv za daljnju analizu koštanih markera pomoću ELISA-e. Tom prilikom će se ektopična kost izvaditi, dio uzoraka će se fiksirati u otopini formalina i pripremiti za snimanje na mikro CT-u i histološku analizu dok će se dio uzoraka pripremiti za genomsku i proteinsku analizu pomoću RNA-Seq, qPCR-a, gel elektroforeze i Western blotting metoda. 2. Model cijeljenja prijeloma. U istraživanju će se koristiti dobro opisani model prijeloma duge kosti u miševa. U pokusu će se proučiti utjecaj BMP3 proteina na proces cijeljenja kosti koji uključuje resorpciju ozlijeđenog koštanog tkiva, stvaranje koštanog kalusa te u konačnici, pregradnju koštanog kalusa i potpunog cijeljenja prijeloma. U pokusnih BMP3-/- i WT miševa starih 2-3 mjeseca, u anesteziji će se napraviti transverzalni zatvoreni prijelom tibije. Grupe životinja će biti uspostavljene prema genotipu i spolu kako bi se mogao izdvojiti utjecaj spola na rezultate. Po završetku pokusa prikupit će se zacijeljene kosti, kontra-lateralne, zdrave kosti kao i uzorak krvi za analizu stanica pomoću protočne citometrije i koštanih markera pomoću ELISA-e. Dio uzoraka će se fiksirati za snimanje na mikro CT-u i histološku analizu, a drugi će se dio uzoraka pripremiti za genomsku i proteinsku analizu pomoću RNA-Seq, qPCR-a, gel elektroforeze i Western blotting metoda. 3. Model osteoporoze. U istraživanju će se koristiti dobro opisani model ovarijektomijom izazvane osteoporoze u miševa. Kod ovarijektomije se operativnim zahvatom uklanjanja jajnika simulira menopauza te će se proučiti utjecaj BMP3 proteina na gubitak kosti zbog nedostatka estrogena u ženki miševa. U postupak će biti uključena i skupina životinja koje će biti lažno operirane (SHAM), a postupak s njima će biti u svemu jednak, osim što se jajnici nakon prikazivanja neće odstraniti. Vrijeme potrebno za razvoj osteoporoze i mjerljiv gubitak kosti iznosi 21 dan, ali će se za pokus i razvoj uznapredovalog stadija osteoporoze životinje držati 42 dana. Po završetku pokusa prikupit će se bedrene kosti, slabinski dio kralješnice kao i uzorak krvi za analizu stanica pomoću protočne citometrije te mjerenje markera koštane pregradnje kao i estrogena pomoću ELISA-e. Dio uzoraka bedrenih kosti će se fiksirati za snimanje na mikro CT-u i histološku analizu, a drugi će se dio uzoraka pripremiti za genomsku i proteinsku analizu pomoću RNA-Seq, qPCR-a, gel elektroforeze i Western blotting metoda. Slabinska kralješnica će biti samo snimljena pomoću mikro CT uređaja.

Experiments will be based on 57BL/6Ntac breed mice with knocked out BMP3 gene (BMP3-/-). In Bmp3-/- mouse a construct containing a β-galactosidase gene reporter, is inserted in the first exon in frame, which creates a non-functional BMP3 protein. As a control, wild type mice of the 57BL/6Ntac breed will be used. Cell culture For in vitro analyses, cell line of mouse pre-osteoblasts, MC3T3, will be used to test activity of purchased BMP3 antibody and protein. In primary cell culture of bone marrow stem cells (BMSC) from Bmp3-/- and WT mice, differentiation, proliferation and maturation of osteoblastic and osteoclastic cells will be investigated, thus exploring the role of BMP3 in these events. Mouse models 1. Ectopic bone formation. The use of full blood as a carrier for BMP6 has proven successful in creating ectopic bone in rats (12). In the experiments, the role BMP3 exerts on the process of de novo bone formation will be studied in subcutaneous ectopic assay. In this assay, human recombinant BMP-6 in the carrier of autologous blood forming a coagulum will be used to assess the osteogenic response in BMP3-/- and WT mice. By adding exogenous BMP3 protein or antibody in the autologous blood coagulum (ABC) with BMP6 additional insights on the role of BMP3 in bone formation will be obtained. The operation will be performed under general anesthesia and the ABC will be implanted in axillar region of the mouse. Time sufficient for bone formation is 14 days after which the experiment is terminated. Animal groups will be established according to combinations of BMP3 proteins, BMP3 antibody and BMP6 protein. Using power analysis, preliminary calculations showed that the groups must have a minimum of 6 members. 5 days and 2 days prior experiment termination fluorescent markers (calcein/tetracyclin) will be injected i.p. in mice for dynamic histomorphometric bone analysis by histology. During experiment termination blood samples will be taken for ELISA analysis. Also ectopic bone samples will be excised, with part of the samples fixed in formalin for micro CT and histology analysis while other part of the samples will be prepared for gene and protein analyses via RNA-Seq, qPCR, gel electrophoresis and Western blotting methods. 2. Bone fracture healing model. In the project, a well-described mouse model of tibial fracture will be used (32). The experiments are designed to assess the role of BMP3 on fracture healing which includes bone resorption of the fractured bone, bone callus formation and bone remodeling and complete fracture healing. In 2-3 months old BMP3-/- i WT mice closed transversal tibial fracture will be introduced under general anesthesia. Prior fracture introduction, through the tibia plateau, a 25G needle will be introduced in the medullar canal. After needle removal through the canal a stainless-steel intramedullary pin will be introduced for bone fragment stabilization. The bone fracture will be introduced 1-2 mm proximally from the distal end of the fibula by using a contraption with weights and dull blade. Bone callus formation will be radiographically monitored every 7 day until day 21 post fracture when the experiment will be terminated. Groups of animals will be established by genotype and gender so that the impact of gender on the results can be extracted. Using power analysis, preliminary calculations showed that the groups must have a minimum of 6 members. 7 days and 3 days prior experiment termination fluorescent markers (calcein/tetracyclin) will be injected i.p. in mice for dynamic histomorphometric bone analysis by histology. During experiment termination blood samples will be taken for ELISA analysis. Also fractured tibia and contralateral healthy tibias will be collected, with part of the samples fixed in formalin for micro CT and histology analysis while other part of the samples will be trimmed to contain bone callus and prepared for gene and protein analyses via RNA-Seq, qPCR, gel electrophoresis and Western blotting methods. 3. Osteoporosis model. In the project a well described model ovariectomy induced osteoporosis will be used (33, 34). In ovariectomy procedure, by the removal of ovaries, menopause is simulated. Lack of estrogen has a profound effect on bone tissue and increase in bone resorption occurs. In the experiment, the role of BMP3 on bone loss due to estrogen deficiency will be examined. Ovariectomy operations will be conducted under general anesthesia where skin will be cut, dorsal peritoneal cavity will be opened and ovaries will be removed. In the procedure will be included a group of falsely operated mice (SHAM), with the same procedure differing only in the lack of ovaries removal, to serve as a negative control. Time to develop osteoporosis is 21 days, although to fully assess the role of BMP3 advanced stage of osteoporosis in mice will be used and the experiment will be terminated on day 42. Only female mice 2 and 4 months old will be used in the experiment. Animal groups will be established according to the genotype. Using the power analysis, preliminary calculations showed that the groups must have a minimum of 8 members. 7 days and 3 days prior experiment termination fluorescent markers (calcein/tetracyclin) will be injected i.p. in mice for dynamic histomorphometric bone analysis by histology. During experiment termination blood samples will be taken for ELISA analysis. Also mice femurs will be collected, with part of the samples fixed in formalin for micro CT and histology analysis (35) while other part of the samples will be trimmed to contain distal part of the femur and prepared for gene and protein analyses via RNA-Seq, qPCR, gel electrophoresis and Western blotting methods.

Ključne riječi

Kost, regeneracija, Bmp3, cijeljenje prijeloma, osteoporoza

Bone, regeneration, Bmp3, fracture repair, osteoporosis

Znanstveno-istraživački projekti

BON3gen

IP-2020-02-5960

25.09.2021

24.09.2025

nije evidentirano

HRK 80.000

Podaci o financiranjima

Podaci o institucijama

Podaci o osobama