J Korean Soc Spine Surg.  2001 Sep;8(3):195-201. 10.4184/jkss.2001.8.3.195.

Responsiveness of Human Intervertebral Disc Cells in Matrix Synthesis To Adenovirus-Mediated Gene Transfer(IGF-1, TGF-beta1 Encoding Genes)

• Lee HM
• Park MS
• Kim H
• Kim HS
• Kang ES
• Kim NH
• Moon SH

• KMID: 2209735
• DOI: http://doi.org/10.4184/jkss.2001.8.3.195

Abstract

BACKGROUND: Genetic modification of cells through gene transfer gains popularity as a sophisticated delivery system in the management of musculoskeletal disease. Anabolic growth factors like IGF-1 BMP-2 OP-1, and TGF-beta1 were candidate for therapeutic purposus for regenerating matrix of intervertebral disc. TGF-beta1, OP-1 and BMP-2 share same pathway i.e. Smad while IGF-1 utilize P13K pathway. Accordingly it is logical to use two cytokine encoding genes which using different signal transduction pathway to upregulate matrix synthesis. PURPOSE: To elucidate the anabolic effect of the combination gene transfer(IGF-1 and TGF-beta1 encoding gene) to human disc cells, cultured in alginate beads.
MATERIALS AND METHODS
Lumbar and cervical intervertebral disc tissue was obtained from surgical disc procedure from fifteen patients. After isolation and culture of the cells, cultures were transduced with first, Adenovirus-TGFbeta1 construct(Ad/TGF-beta1) and Ad/IGF-1 respectively, second, with combination of two viral constructs(Ad/TGF-beta1+Ad/IGF-1). Cultures treated with saline and Ad/luciferase served as control. Then cultures were incorporated into alginate beads. Exogenous TGF-beta1(2ng/ml) and IGF-1(100ng/ml) were administered also. Newly synthesized proteoglycan was assessed using S35 incorporation using chromatography on Sephadex G-25 in PD-10 column.
RESULTS
In cultures transduced with single therapeutic gene construct, there were statistically significant 2.9 fold(Ad/TGF-beta1 and 1.8 fold(Ad/IGF-1) increase in newly synthesized proteoglycan comparing control(p<0.05). In culture transduced with double combination of therapeutic gene construct, there were 3.9 fold(Ad/TGF-beta1+Ad/IGF-1) increase in newly synthesized proteo-glycan comparing control(p<0.05). Culture treated with TGF-beta1(2ng/ml) showed 3.9 fold increase and IGF-1(100ng/ml) 2.9 fold increase, TGF-beta1+IGF-1 4.2 fold increase in proteoglycan synthesis compared to control.
CONCLUSIONS
Combination gene therapy provided efficient mechanism of upregulating matrix regeneration of the human inter-vertebral disc cells.

Keyword

Combination gene therapy; Intervertebral disc; Proteoglycan; TGF-beta1; IGF-1

MeSH Terms

Anabolic Agents
Cells, Cultured
Chromatography
Genetic Therapy
Humans*
Insulin-Like Growth Factor I
Intercellular Signaling Peptides and Proteins
Intervertebral Disc*
Logic
Musculoskeletal Diseases
Proteoglycans
Regeneration
Signal Transduction
Transforming Growth Factor beta1*
Anabolic Agents
Insulin-Like Growth Factor I
Intercellular Signaling Peptides and Proteins
Proteoglycans
Transforming Growth Factor beta1

Figure

• 

  Fig. 1. Newly synthesized proteoglycan in three dimensional culture of human intervertebral disc cells presented as percent control. Adenovirus-TGF-beta1 construct(Ad/ TGF-ß1) with an MOI of 75 showed 2.9 fold increase, adenovirus-IGF-1 construct(Ad/IGF-1) with an MOI of 75 showed 1.8 fold increase, Ad/TGF-ß1+AdIGF-1 with an MOI of 75 showed 3.9 fold increase in newly synthesized proteoglycan compared to saline and viral control. *:p<0.05

• 

  Fig. 2. Newly synthesized proteoglycan in three dimensional culture of human intervertebral disc cells presented as percent control. TGF-ß1(2 ng/ml) showed 3.9 fold increase, IGF-1(100 ng/ml) showed 2.9 fold increase, and TGF-ß1(2 ng/ml) + IGF-1(100 ng/ml) showed 4.2 fold increase in newly synthesized proteoglycan compared to saline and viral control. *:p<0.05

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