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ZJUS-B
Journal of Zhejiang University-SCIENCE B (Biomedicine & Biotechnology)  2016, Vol. 17 Issue (1): 30-42    DOI: 10.1631/jzus.B1500182
Article     
Adenovirus-mediated GDF-5 promotes the extracellular matrix expression in degenerative nucleus pulposus cells
Xu-wei Luo,Kang Liu,Zhu Chen,Ming Zhao,Xiao-wei Han,Yi-guang Bai,Gang Feng()
Research Institute of Tissue Engineering and Stem Cells, Nanchong Central Hospital and the Second Clinical Institute of North Sichuan Medical College, Nanchong 637000, China
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Abstract  

Objective: To construct a recombinant adenovirus vector-carrying human growth and differentiation factor-5 (GDF-5) gene, investigate the biological effects of adenovirus-mediated GDF-5 (Ad-GDF-5) on extracellular matrix (ECM) expression in human degenerative disc nucleus pulposus (NP) cells, and explore a candidate gene therapy method for intervertebral disc degeneration (IDD). Methods: Human NP cells of a degenerative disc were isolated, cultured, and infected with Ad-GDF-5 using the AdEasy-1 adenovirus vector system. On Days 3, 7, 14, and 21, the contents of the sulfated glycosaminoglycan (sGAG), deoxyribonucleic acid (DNA) and hydroxyproline (Hyp), synthesis of proteoglycan and collagen II, gene expression of collagen II and aggrecan, and NP cell proliferation were assessed. Results: The adenovirus was an effective vehicle for gene delivery with prolonged expression of GDF-5. Biochemical analysis revealed increased sGAG and Hyp contents in human NP cells infected by Ad-GDF-5 whereas there was no conspicuous change in basal medium (BM) or Ad-green fluorescent protein (GFP) groups. Only cells in the Ad-GDF-5 group promoted the production of ECM, as demonstrated by the secretion of proteoglycan and up-regulation of collagen II and aggrecan at both protein and mRNA levels. The NP cell proliferation was significantly promoted. Conclusions: The data suggest that Ad-GDF-5 gene therapy is a potential treatment for IDD, which restores the functions of degenerative intervertebral disc through enhancing the ECM production of human NP cells.

Key words： Intervertebral disc      Degeneration      Growth and differentiation factor-5 (GDF-5)      Adenovirus      Gene therapy      Nucleus pulposus     
Received: 30 July 2015      Published: 01 January 2016
Fund:  Project supported by the National Natural Science Foundation of China(Nos. 81171472, 81201407);the Innovation Team Project of Sichuan Provincial Education Department(No. 13TD0030);the Major Transformation Cultivation Project of Sichuan Provincial Education Department(No. 15CZ0021);the Science and Technology Project of Nanchong City, China(No. 14A0021)
Corresponding Authors: Gang Feng     E-mail: fenggangncch@163.com
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Xu-wei Luo
Kang Liu
Zhu Chen
Ming Zhao
Xiao-wei Han
Yi-guang Bai
Gang Feng
Cite this article:

Xu-wei Luo,Kang Liu,Zhu Chen,Ming Zhao,Xiao-wei Han,Yi-guang Bai,Gang Feng. Adenovirus-mediated GDF-5 promotes the extracellular matrix expression in degenerative nucleus pulposus cells. Journal of Zhejiang University-SCIENCE B (Biomedicine & Biotechnology), 2016, 17(1): 30-42.

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http://www.zjujournals.com/xueshu/zjus-b/10.1631/jzus.B1500182     OR     http://www.zjujournals.com/xueshu/zjus-b/Y2016/V17/I1/30

Molecule Primer sequence Product size (bp)
?Collagen II 5'-TCCCAGAACATCACCTACC-3' (sense) 131
? 5'-AACCTGCTATTGCCCTCT-3' (antisense)
?Aggrecan 5'-CGCTACTCGCTGACCTTT-3' (sense) 106
? 5'-GCTCATAGCCTGCTTCGT-3' (antisense)
?β-Actin 5'-GAGCTACGAGCTGCCTGACG-3' (sense) 128
? 5'-GTAGTTTCGTGGATGCCACAG-3' (antisense)
Table 1 Primer sequences used for RT-PCR
Fig. 1 Western blotting results Proteins were electrophoretically separated on 10% criterion separation SDS-polyacrylamide gel on a mini-protean 3 system apparatus. Western blotting was performed with goat anti-GDF-5 polyclonal antibody. (a) Western blotting of GDF-5 recovered from the supernatant of cultured HEK293 cells at the second day post-infection; (b) Western blotting of GDF-5 recovered from the supernatant of NP cells at 21 d post-infection. Lane 1: control group; Lane 2: Ad-GFP group; Lane 3: Ad-GDF-5 group
Fig. 2 Flow cytometry identification result of NP cells
Fig. 3 sGAG/DNA ratio for human NP cells treated with Ad-GDF-5 The sGAG and DNA expression levels were measured in NP cells that were infected with increasing multiplicity of infection (MOI) ratios of Ad-GDF-5. Human NP cells were collected at 3, 7, and 14 d after the primal transduction, and the sGAG and DNA levels were detected using the DMMB colorimetric assay and the Hoechst 33258 DNA quantitation kit, respectively. Data points are presented as mean±SD (n=3). * P<0.05 vs. MOI 50; ** P<0.01 vs. control; ## P<0.01 vs. MOI 25
Fig. 4 Levels of sulfated glycosaminoglycan (sGAG) and hydroxyproline (Hyp) normalized to DNA content in NP cells (a) The sGAG/DNA ratio in NP cells infected by Ad-GDF-5, Ad-GFP, or no adenovirus (BM group); (b) The Hyp/DNA ratio in NP cells infected with Ad-GDF-5, Ad-GFP, or no adenovirus (BM group). Data points are presented as mean±SD (n=3). * P<0.05, ** P<0.01, vs. BM group; # P<0.05, ## P<0.01, vs. Ad-GFP group
Fig. 5 Safranin-O staining (red) of the proteoglycan-rich ECM of human NP cells NP cells were stained at 3, 7, 14, and 21 d after being infected by Ad-GDF-5, Ad-GFP, or no adenovirus (BM group). Scale bar=50 Μm (Note: for interpretation of the references to color in this figure legend, the reader is referred to the web version of this article)
Fig. 6 Immunostaining of human NP cells for type-II collagen and aggrecan after they were infected by Ad-GDF-5, Ad-GFP, or no adenovirus (BM group) Type-II collagen is stained dark brown (a), and aggrecan is shown by red fluorescent staining (b). Scale bar=50 Μm (Note: for interpretation of the references to color in this figure legend, the reader is referred to the web version of this article)
Fig. 7 Concentrations of collagen II (Col II) and aggrecan in NP cells at 21th days post-infection by ELISA Data points are presented as mean±SD (n=3). * P<0.05 vs. BM group; # P<0.05 vs. Ad-GFP group
Fig. 8 Gene expression patterns in human NP cells following infection by Ad-GDF-5, Ad-GFP, or no adenovirus (BM group) over 3, 7, 14, and 21 d (a) Collagen II mRNA levels; (b) Aggrecan mRNA levels. Data points are presented as mean±SD (n=6). * P<0.05, ** P<0.01, vs. BM group; # P<0.05, ## P<0.01, vs. Ad-GFP group
Fig. 9 NP cell proliferation after transfection Data points are presented as mean±SD (n=3). ** P<0.01 vs. BM group; ## P<0.01 vs. Ad-GFP group
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