Optimal Construction and Delivery of Dual-Functioning Lentiviral Vectors for Type I Collagen-Suppressed Chondrogenesis in Synovium-Derived Mesenchymal Stem Cells
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This study aims to deliver both transforming growth factor β3 (TGF-β3) and shRNA targeting type I collagen (Col I) by optimal construction and application of various dual-functioning lentiviral vectors to induce Col I-suppressed chondrogenesis in synovium-derived mesenchymal stem cells (SMSCs).
We constructed four lentiviral vectors (LV-1, LV-2, LV-3 and LV-4) with various arrangements of the two expression cassettes in different positions and orientations. Col I inhibition efficiency and chondrogenic markers were assessed with qPCR, ELISA and staining techniques. Among the four vectors, LV-1 has two distant and reversely oriented cassettes, LV-2 has two distant and same-oriented cassettes, LV-3 has two proximal and reversely oriented cassettes, and LV-4 has two proximal and same-oriented cassettes. Col I and chondrogenic markers, including type II collagen (Col II), aggrecan and glycosaminoglycan (GAG), were examined in SMSCs cultured in 3-D alginate hydrogel.
All of the four vectors showed distinct effects in Col I level as well as diverse inductive efficiencies in upregulation of the cartilaginous markers. Based on real-time PCR results, LV-1 was optimal towards Col I-suppressed chondrogenesis.
LV-1 vector is competent to promote Col I-suppressed chondrogenesis in SMSCs.
KEY WORDSdual-functioning lentiviral vector synovial mesenchymal stem cells (SMSCs) TGF-β3 type I collagen chondrogenesis
- Col I
type I collagen
- Col II
type II collagen
recombinant lentiviral vector that expresses both shRNA targeting type I collagen and TGF- β3
recombinant lentiviral vector that specifically expresses TGF-β3
short hairpin RNA
synovium-derived mesenchymal stem cell
transforming growth factor-β3
This research was financially supported by AcRF Tier 1 Grant RG64/08, Ministry of Education (MoE), and NMRC/EDG/1001/2010, Singapore.
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