The Role of RING Ubiquitin Ligases in Biologic and Oncologic Processes in Tissues of Mesenchymal Origin (RING UB LIGASE)
Stage 1:
Bone marrow will be collected from otherwise healthy patients undergoing orthopedic surgery - arthroplasty for the treatment of degenerative joint disease or traumatised patients. The bone marrow will be collected from disposable tissue that is removed during the normal sequence of the surgery. These cells will be used for:
Establishment of the role of RING ubiquitin ligases in osteogenic progenitors proliferation and differentiation in culture.
I. To determine the levels of RING ubiquitin ligases mRNA and protein in differentiating human mesenchymal precursor cells: To test the correlation between levels of RING ubiquitin ligases and degree of osteogenic differentiation, we will extract mesenchymal precursor cells from the collected tissue and test the mRNA protein level of RING ubiquitin ligases upon induction of differentiation. Specifically, we will compare the levels of RING ubiquitin ligases before and after the initiation of differentiational stimulus in time dependent manner by western blot and real time PCR analysis.
II. To test the impact of specific RING ubiquitin ligases on differentiation of human mesenchymal precursor cells: to test the impact of RING ubiquitin ligases on the differentiation of mesenchymal precursor cell from the collected tissue by testing the expression of classical differentiational bone markers by flow cytometry (fibronectin, CD105) and by Alkaline phosphatase (ALP) activity assay. For this aim we developed both a constitutive and Dox-regulated conditional overexpression and shRNA lenti-viral systems that enables efficient modulation of these RING ubiquitin ligases level.
III. Determine the role of RING ubiquitin ligases in proliferation and survival of human mesenchymal precursor cells: Via inhibition or overexpression of ligases in mesenchymal progenitor cells we will test the role of these ligases in proliferation and survival of mesenchymal precursors by using MTT assay, Propidion-Iodid (PI) and tunnel assays in flow cytometry analysis.
Stage 2:
Collection of connective tissue from patients with malignancies of musculoskeletal origin. The tissue that will be used is part of the resected tumor specimens. The tissue will be used for:
The establishment of the role/s of RING ligases in musculoskeletal cancers using cell culture and in vivo activation. Test if the expression of selected positive candidates from stage 1 correlates with cancer development and progression in human-derived samples Independently of our mechanistic experiment we aim to determine the relevance of these ligases to human musculoskeletal cancers. As the first step we will screen primary tumor biopsies at the protein level correlates with cancer grade and prognosis. Toward this aim we recently generated a highly specific several anti-monoclonal antibodies in our laboratory as well use comercial available antibodies .
Aperçu de l'étude
Statut
Description détaillée
Stage 1:
Bone marrow will be collected from otherwise healthy patients undergoing orthopedic surgery - arthroplasty for the treatment of degenerative joint disease or traumatised patients. The bone marrow will be collected from disposable tissue that is removed during the normal sequence of the surgery. These cells will be used for:
Establishment of the role of RING ubiquitin ligases in osteogenic progenitors proliferation and differentiation in culture.
I. To determine the levels of RING ubiquitin ligases mRNA and protein in differentiating human mesenchymal precursor cells: To test the correlation between levels of RING ubiquitin ligases and degree of osteogenic differentiation, we will extract mesenchymal precursor cells from the collected tissue and test the mRNA protein level of RING ubiquitin ligases upon induction of differentiation. Specifically, we will compare the levels of RING ubiquitin ligases RING ubiquitin ligases before and after the initiation of differentiational stimulus in time dependent manner by western blot and real time PCR analysis.
II. To test the impact of RING ubiquitin ligases on differentiation of human mesenchymal precursor cells: to test the impact of RING ubiquitin ligases on the differentiation of mesenchymal precursor cell from the collected tissue by testing the expression of classical differentiational bone markers by flow cytometry (fibronectin, CD105) and by Alkaline phosphatase (ALP) activity assay. For this aim we developed both a constitutive and Dox-regulated conditional RING ubiquitin ligases overexpression and shRNA lenti-viral systems that enables efficient modulation of RING ubiquitin ligases level.
III. Determine the role of RING ubiquitin ligases in proliferation and survival of human mesenchymal precursor cells: We will test the role of their in proliferation and survival of mesenchymal precursors by using MTT assay, Propidion-Iodid (PI) and tunnel assays in flow cytometry analysis.
Stage 2:
Collection of connective tissue from patients with malignancies of musculoskeletal origin. The tissue that will be used is part of the resected tumor specimens. The tissue will be used for:
The establishment of the role/s of RING ubiquitin ligases in musculoskeletal cancers using cell culture and in vivo.
I. Test the role of our in proliferation and survival in musculoskeletal cancers: Using our lentiviral systems we will test the biological impact of inhibition and overexpression in musculoskeletal neoplastic cells.
II. Test whether inhibition or overexpression of these ligases impacts the biology of musculoskeletal cancers.We will check if inhibition of these ligases in malignant cells of musculoskeletal origin by lenti-viral infections induces differentiation. Infected cells will be analyzed for expression of differentiational markers in flow cytometry and immunohistochemistry.
III. Test if expression of RING ubiquitin ligases correlates with cancer development and progression in human-derived samples Independently of our mechanistic experiment we aim to determine the relevance of RING ubiquitin ligases to human musculoskeletal cancers.
Patients consent will be signed after properly informed at time of pre-surgical preparation visit. In cases when pre-surgical visit is not held consent will be signed prior to the day of surgery.
Type d'étude
Inscription (Anticipé)
Contacts et emplacements
Lieux d'étude
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Haifa, Israël
- Recrutement
- Rambam MC
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Contact:
- Rostislav Novak, MD/PhD
- Numéro de téléphone: +972502061188
- E-mail: n_rosti@rambam.health.gov.il
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Contact:
- Amir M Orian, MD/PhD
- Numéro de téléphone: +97248295448/9
- E-mail: mdoryan@tx.technion.ac.il
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Chercheur principal:
- David Nikomarov, MD
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Sous-enquêteur:
- Rostislav Novak, MD/PhD
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Sous-enquêteur:
- Amir Oryan (Orian), MD/PhD
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Critères de participation
Critère d'éligibilité
Âges éligibles pour étudier
Accepte les volontaires sains
Sexes éligibles pour l'étude
Méthode d'échantillonnage
Population étudiée
- Healthy patients undergoing orthopedic surgery - arthroplasty for the treatment of degenerative joint disease or traumatised patients.
- Patients with malignancies of musculoskeletal origin.
La description
Group 1:
- Inclusion Criteria:
- Healthy patients undergoing orthopedic surgery - arthroplasty for the treatment of degenerative joint disease or traumatised patients.
- Exclusion Criteria:
- Any malignancy in the past or active infection.
Group 2:
- Inclusion Criteria:
- patients with malignancies of musculoskeletal origin.
- Exclusion Criteria:
- Any malignancy of unknown origin or malignancy of multiple origins or active infection.
Plan d'étude
Comment l'étude est-elle conçue ?
Détails de conception
Cohortes et interventions
Groupe / Cohorte |
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The role of RING ubiquitin ligases in osteogenic progenitors
Bone marrow (BM) will be collected from healthy patients undergoing orthopedic surgery.
BM will be collected from disposable tissue that is removed during the normal sequence of the surgery.
|
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The role of RING ubiquitin ligases in musculoskeletal cancer
Collection of connective tissue from patients with tumors of musculoskeletal origin - a part of the resected tumor specimens.
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Que mesure l'étude ?
Principaux critères de jugement
Mesure des résultats |
Description de la mesure |
Délai |
|---|---|---|
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Protein and mRNA levels of RING ubiquitin ligases during osteogenesis
Délai: 5 years
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To determine the levels of RING ubiquitin ligases mRNA and protein in differentiating human mesenchymal precursor cells.
We will extract mesenchymal precursor cells from the collected tissue and test the mRNA and protein levels of RING ubiquitin ligases upon induction of differentiation.
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5 years
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Protein and mRNA levels of RING ubiquitin ligases in musculoskeletal tumors and its correlation to staging and grading
Délai: Time Frame: * 5 years
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It is not clear if RING ubiquitin ligases protein levels are elevated in soft tissue-derived cancers (primary and metastatic tumors) and whether there is a correlation between RING ubiquitin ligases levels and staging and grading of soft tissue-derived cancers.
For this reason, we will check whether RING ubiquitin ligases expression correlates with cancer development and progression in human-derived samples.
We will determine the relevance of RING ubiquitin ligases to human musculoskeletal cancers.
To do this we will screen primary tumor biopsies for RNA and protein levels of RING ubiquitin ligases .
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Time Frame: * 5 years
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Collaborateurs et enquêteurs
Parrainer
Collaborateurs
Dates d'enregistrement des études
Dates principales de l'étude
Début de l'étude
Achèvement primaire (Anticipé)
Achèvement de l'étude (Anticipé)
Dates d'inscription aux études
Première soumission
Première soumission répondant aux critères de contrôle qualité
Première publication (Estimation)
Mises à jour des dossiers d'étude
Dernière mise à jour publiée (Estimation)
Dernière mise à jour soumise répondant aux critères de contrôle qualité
Dernière vérification
Plus d'information
Termes liés à cette étude
Autres numéros d'identification d'étude
- 0180-14-RMB
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