J Korean Soc Spine Surg.  2001 Mar;8(1):1-7.

Adenovirus-Mediated Therapeutic Gene Transfer: Matrix Synthesis of Human Intervertebral Disc Cells

• Moon SH
• Park MS
• Park JO
• Lee HM
• Seo YJ
• Kim NH
• Kang ES

Abstract

STUDY DESIGN: In vitro experiment to determine the matrix synthesis of human intervertebral disc (IVD) cell to adenovirus-mediated therapeutic gene transfer.
OBJECTIVES
To elucidate proteoglycan and collagen synthesis of human IVD cells in vitro to adenovirus-mediated transfer of cDNA of transforming growth factor-beta 1(TGF-beta). SUMMARY OF LITERATURE REVIEW: Sophisticated method to delivery of growth factors, in continuous manner, is the genetic modi-fication of disc cells through gene transfer. Confirming susceptibility of human IVD cell to adenovirus, anabolic response of human IVD cells to therapeutic gene transfer should be next step.
MATERIALS AND METHODS
IVD tissue was obtained from fourteen patients with grade III, IV degeneration. Isolation and culture of disc cells were performed. Disc cells were treated with either Ad/TGF-beta exogenous TGF-beta. Control cultures were treated with either saline or Ad/luciferase. Newly synthesized proteoglycans were assessed by 35 S-sulfate incorporation using chro-matography on Sepadex G-25 in PD-10 columns. Uptake of 3 H proline was used to measure synthesis of collagen and noncol-lagen protein.
RESULTS
Culture treated with Ad/TGF-beta showed 3 fold increase in proteoglycan synthesis (p<0.05), culture with exogenous T G F -beta failed to demonstrate increase in proteoglycan synthesis. In collagen and noncollagen synthesis, cultures with Ad/TGF-beta and exogenous TGF-beta showed similar 3.7 fold increase in collagen and 2.7 fold increase in noncollagen synthesis comparing control (p<0.05).
CONCLUSION
Adenovirus-mediated gene transfer appears to be an efficient technique for modulating biologic activity of human intervertebral disc cells in terms of matrix synthesis.

Keyword

Gene Transfer; Ad/TGF-beta; Proteoglycan; Collagen

MeSH Terms

Adenoviridae
Collagen
DNA, Complementary
Humans*
Intercellular Signaling Peptides and Proteins
Intervertebral Disc*
Proline
Proteoglycans
Transforming Growth Factor beta
Collagen
DNA, Complementary
Intercellular Signaling Peptides and Proteins
Proline
Proteoglycans
Transforming Growth Factor beta

Figure

• 

  Fig. 1. Content of newly synthesized proteoglycan as assayed by incorporation of 35S-sulfate. Human intervertebral disc cells transduced by adenovirus-TGF β1 construct(150 MOI) showed 3 fold increase in newly synthesized proteoglycan compared to those treated with normal saline∗(p<0.05), while culture treated by TGF-β1(2 ng/ml) showed no increase in newly synthesized proteoglycan compared to those treated with normal saline(p=0.07).

• 

  Fig. 2. Content of newly synthesized collagen and noncollagen as assayed by incorporation of 3H-proline. Human inter-vertebral disc cells transduced by adenovirus-TGFβ1 construct(150 MOI) and thoses treated by TGF-β1 (2 ng/ml) showed 3.5 fold increase in collahen synthesis and 2.5 fold increase in noncollagen synthesis∗(p<0.05).

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