Transgenic viral vector mediated delivery of stromal cell-derived factor 1α and suprresion of type I collagen by RNA interference for enhanced tissue regeneration

• Main Author: Fang, Yu
• Other Authors: Wang Dongan
• Format: Final Year Project
• Language: English
• Published: 2012
• Subjects: DRNTU::Engineering::Chemical engineering::Biotechnology; DRNTU::Science::Medicine::Tissue engineering
• Online Access: http://hdl.handle.net/10356/50127
• Institution: Nanyang Technological University

In tissue engineering, implanted graft will integrate with the peripheral tissue and promote the structural and functional restoration of the tissue. In the first part stromal cell-derived factor (SDF) 1α was studied, which is a chemokine that can promote the chemotactic migration of stem/progenitor cells to the injured site for regeneration. Therefore, in this project, an adenoviral vector that can mediate the expression of SDF-1α (Ad-SDF) in the target cells was constructed. As target cells, chondrocytes were transduced with Ad-SDF in vitro. Subsequently, SDF expression and production level increased, as monitored by ELISA and RT-PCR. Alternatively, living scaffold (LS) fabricated with live chondrocytes was transduced with Ad-SDF for transplantation into nude mice. After transduction, LS expressed higher concentrations of SDF-1α, and a gradient of SDF-1α was therefore created in the circulated blood. By investigating blood and implant cell samples with fluorescence-activated cell sorting (FACS) for CXCR (SDF-1α receptor) positive cells, the hematopoietic stem cells and mesenchymal stem cells were observed to migrate against the gradient to the LS transplantation site. As a result, the recruit of the stem cells and progenitor cells enhanced the regenerative capabilities of the transplanted tissue; this was confirmed by in vivo immunohistochemistry staining. In the second part, a dual functioning lentiviral vector LV-D was constructed to express TGF-β3 and type I collagen (Col I) suppressing shRNA. TGF-β3 can potentially induce the differentiation of mesenchymal stem cells into chondrocytes and enhance tissue regeneration. Despite that, the introduction of TGF-β3 will induce the upregulation of Col I, which results in formation of fibrous cartilage. Therefore, RNA interference technology was used to suppress the expression of Col I by degrading its mRNA. Based on this vector LV-D, a set of protocols for transduction was established and optimized. To aid the transduction, polybrene was added to attenuate the repulsive charges present on cell surface, the most favorable polybrene concentration was determined. A modified ELISA method for measuring TGF-β3 without hydrochloric acid activation was formulated as well to find out the relationship between total TGF-β3 and activated TGF-β3.