Lentiviral vector-mediated shRNAs targeting a functional isoform of the leptin receptor (Ob-Rb) inhibit cartilage degeneration in a rat model of osteoarthritis

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Presentation transcript:

Lentiviral vector-mediated shRNAs targeting a functional isoform of the leptin receptor (Ob-Rb) inhibit cartilage degeneration in a rat model of osteoarthritis S.L. Deng, J. Zhu, Q. Huang, W.L. Fu, Q. Li, G. Chen, X. Tang, J. Li Osteoarthritis and Cartilage Volume 25, Issue 11, Pages 1912-1921 (November 2017) DOI: 10.1016/j.joca.2017.08.003 Copyright © 2017 Osteoarthritis Research Society International Terms and Conditions

Fig. 1 (A) DNA sequencing confirmed that the shRNA3 sequences had inserted correctly into the pGC-LV shuttle plasmids. (B) Optimization of the transfection conditions. Representative examples of flow cytometry and confocal microscopy measurements showing that the transfection efficiency is 86.41% under the optimal transfection condition (Enhanced Infection Solution with 30 multiplicity of infection (MOI) lentiviral vectors). (C) Selection of optimal LV-shRNA. qRT-PCR (left) and western blotting (right) analysis showed that LV-shRNA3 achieved the highest silencing efficiency, as its efficiency was greater than that of the LV-NTCs; therefore, LV-shRNA3 was used for the subsequent experiment. Significant differences are indicated. RQ, relative quantity. Osteoarthritis and Cartilage 2017 25, 1912-1921DOI: (10.1016/j.joca.2017.08.003) Copyright © 2017 Osteoarthritis Research Society International Terms and Conditions

Fig. 2 In vitro Ob-Rb downregulation influenced the mRNA expression levels of pro-inflammatory (TNF-α, IL-1β and IL-6), anabolic (type II collagen) and catabolic (ADAMTS-4, ADAMTS-5, MMP-9, COX-2 and NOS-2) mediators. Relative expression levels (means with 95% confidence intervals (CIs)) in the LV-shRNA3 and Normal groups compared to the LV-NTC group were calculated using the comparative CT (2-△△CT) method. Significant differences are indicated. RQ, relative quantity. Osteoarthritis and Cartilage 2017 25, 1912-1921DOI: (10.1016/j.joca.2017.08.003) Copyright © 2017 Osteoarthritis Research Society International Terms and Conditions

Fig. 3 Representative western blotting bands for ADAMTS-5, MMP-9 and type II collagen. These results were consistent with the corresponding qRT-PCR results indicated in Fig. 2. Osteoarthritis and Cartilage 2017 25, 1912-1921DOI: (10.1016/j.joca.2017.08.003) Copyright © 2017 Osteoarthritis Research Society International Terms and Conditions

Fig. 4 Comparisons of cartilage histological findings and Osteoarthritis Research Society International (OARSI) scores among the groups at 2, 4, 8 and 12 weeks post-transfection. Sham, sham operation with PBS injection; OA, osteoarthritis model with PBS injection; OA + LV-NTC, osteoarthritis model with lentiviral vector-mediated non-targeting control sequence injection; OA + LV-shRNA3, osteoarthritis model with lentiviral vector-mediated Ob-Rb shRNA3 injection. (A) Representative sections stained with toluidine blue are shown at 5× magnification. ac – articular cartilage; sd – surface discontinuity; er – erosion; den – denudation; def – deformation. Scale bar, 200 μm. (B) Comparison of OARSI scores among the groups at 2, 4, 8 and 12 weeks post-transfection. Data are presented as means with 95% CIs. *P ≤ 0.001 vs OA; #P < 0.05 vs OA. Osteoarthritis and Cartilage 2017 25, 1912-1921DOI: (10.1016/j.joca.2017.08.003) Copyright © 2017 Osteoarthritis Research Society International Terms and Conditions

Fig. 5 Comparison of immunohistochemical staining and semi-quantitative analysis results among the groups at 4 weeks post-transfection. Sham, sham operation with PBS injection; OA, osteoarthritis model with PBS injection; OA + LV-NTC, osteoarthritis model with lentiviral vector-mediated non-targeting control sequence injection; OA + LV-shRNA3, osteoarthritis model with lentiviral vector-mediated Ob-Rb shRNA3 injection. (A) Representative sections subjected to immunohistochemical staining for Ob-Rb and ADAMTS-5, MMP-9 and type II collagen expression (×400). Immunolabeled chondrocytes (arrow). (B) Semi-quantitative analysis. The proportions of chondrocytes that stained positive for Ob-Rb, ADAMTS-5 and MMP-9 were calculated and expressed as the percentages of positively stained cells within each region. Data are presented as means with 95% CIs. Significant differences vs OA group are indicated. Osteoarthritis and Cartilage 2017 25, 1912-1921DOI: (10.1016/j.joca.2017.08.003) Copyright © 2017 Osteoarthritis Research Society International Terms and Conditions