Thesis Format
Monograph
Degree
Master of Science
Program
Physiology and Pharmacology
Collaborative Specialization
Musculoskeletal Health Research
Supervisor
Grol, Matthew W.
Abstract
Rationale: Osteoarthritis (OA) features cartilage loss, bone remodeling, and chronic inflammation. Dysregulated transforming growth factor beta-1 (TGF-β1) in OA and its constitutive overexpression risks unwanted tissue growth. We aimed to develop an inflammation-inducible TGF-β1 delivery system using cell culture models.
Hypothesis: An inducible promoter will provide controlled, context-dependent Tgfb1 expression with fewer adverse effects than a constitutive promoter.
Methods: We cloned Tgfb1 with either endothelial leukocyte adhesion molecule-1 (Elam1) inducible promoter or elongation factor 1-alpha (Ef1a) constitutive promoter into plasmids, transfected them into C3H10T1/2 cells, and assessed Tgfb1 expression using qPCR. Expression downstream of Ef1a was sustained while expression downstream of Elam1 was induced by lipopolysaccharide (LPS).
Results: Ef1a drove robust Tgfb1 expression, but the Elam1 failed to drive expression in response to LPS in this cell line.
Conclusion: Ef1a promoter effectively drives constitutive Tgfb1 overexpression in C3H10T1/2 cells; further Elam1 studies are required.
Significance: This work supports the potential for inflammation-inducible TGF-β1 expression to protect chondrocytes in OA, guiding future in-vivo studies.
Summary for Lay Audience
Background: Osteoarthritis (OA) is a painful and debilitating joint condition that leads to the breakdown of cartilage and bone. Unfortunately, there are currently no treatments that can stop OA from getting worse. One of the reasons OA is so tricky to manage is that the balance of important growth factors, like TGF-β1, is often disrupted. While TGF-β1 is crucial for keeping our joints healthy, too much of it can cause problems by making tissues grow where we don’t want them to.
Research approach: To address this challenge, we were exploring a new way to deliver TGF-β1 that only activated when it was needed—in other words, when there was inflammation. We designed two types of genetic constructs: one that produced Tgfb1 all the time and another that turned on Tgfb1 production only in response to inflammation. We tested these constructs in lab-grown cells that were commonly used to study joint diseases. To see if our inflammation-triggered construct would work, we used a substance called lipopolysaccharide (LPS) to induce the kind of inflammation seen in OA.
Results: The construct designed to produce Tgfb1 continuously worked well, just as we expected, and the cells produced high levels of Tgfb1. However, the inflammation-activated construct did not produce much Tgfb1, even after we tried to trigger it with LPS. This leaves us unsure if the construct was working under the conditions we used in our tests.
Next Steps: These results tell us that we need to do more research, possibly using conditions that are closer to what happens in OA. If we can get this inflammation-activated approach to work, it could lead to new treatments that target only the affected areas in OA, reducing side effects and helping patients more effectively.
Recommended Citation
Thomas, Anisha, "Inflammation-inducible Strategies for Growth Factor Gene Therapy to Promote Joint Tissue Repair: Applications for Osteoarthritis Therapy" (2024). Electronic Thesis and Dissertation Repository. 10481.
https://ir.lib.uwo.ca/etd/10481