Group 1: Trauma Operation for Otherwise Healthy Patients Clinical Trial
Official title:
The Role of RING Ubiquitin Ligases in Biologic and Oncologic Processes in Tissues of Mesenchymal Origin
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 .
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.
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Observational Model: Cohort