Combined expression of CTGF and tissue inhibitor of metalloprotease-1 promotes synthesis of proteoglycan and collagen type II in rhesus monkey lumbar intervertebral disc cells in vitro.
BACKGROUND
Low back pain has emerged as a widespread disease often caused by intervertebral disc degeneration. This study aimed to establish an in vitro cell culture model of rhesus monkey lumbar intervertebral discs and to investigate the effect of combined connective tissue growth factor (CTGF) and tissue inhibitor of metalloprotease-1 (TIMP-1) expression mediated by adeno-associated virus (AAV) on collagen type II and proteoglycan levels. The purpose of these investigations was to explore potential methods for relieving the degeneration of lumbar intervertebral disc cells.


METHODS
Rhesus monkey lumbar intervertebral disc nucleus pulposus cells (NPCs) were isolated by enzyme digestion, cultured, and transduced with rAAV2-CTGF-IRES-TIMP-1, rAAV2-CTGF, or rAAV2-TIMP-1 at a multiplicity of infection (MOI) of 10(6). The expression of collagen type II and proteoglycan was measured using RT-PCR and Western blotting. The synthetic rate of proteoglycan was measured using (35)S incorporation.


RESULTS
Rhesus monkey lumbar intervertebral disc NPCs were transduced with rAAV2-CTGF-IRES-TIMP-1, rAAV2-CTGF, and rAAV2-TIMP-1 and the transduced genes were expressed and detected. Compared to the control, CTGF promoted the synthesis of collagen type II and proteoglycan. TIMP-1 showed an enhancing effect on the expression of proteoglycan but no effect on collagen type II. Expression of both genes in rhesus monkey lumbar intervertebral disc NPCs significantly enhances the synthesis of proteoglycan and collagen type II.


CONCLUSIONS
Single gene transduction of CTGF or TIMP-1 can enhanced synthesis of proteoglycan. CTGF expression can also enhance collagen type II protein synthesis. Combined transduction of both CTGF and TIMP1 can significantly promote the expression of proteoglycan and collagen type II to levels greater than transduction of a single gene alone. Our study provides a good basis for multi-gene therapy to treat lumbar intervertebral disc degeneration.
Liu
Yong
Y
Department of Orthopaedic Surgery, Affiliated Hospital of Medical College, Qingdao University, Qingdao, Shandong 266003, China. liuyong@medmail.com.cn
Kong
Jie
J
Chen
Bo-hua
BH
Hu
You-gu
YG
D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D024043:Collagen Type II / Q000096:biosynthesis*; D055513:Connective Tissue Growth Factor / Q000235:genetics / Q000378:metabolism*; D007403:Intervertebral Disc / Q000166:cytology*; D008253:Macaca mulatta; D011509:Proteoglycans / Q000096:biosynthesis*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000235:genetics / Q000378:metabolism*; D014161:Transduction, Genetic
D024043:Collagen Type II; D011509:Proteoglycans; D019715:Tissue Inhibitor of Metalloproteinase-1; D055513:Connective Tissue Growth Factor