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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 factorbeta 1(TGF- β1).
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 A d/TGF- β1exogenous TGF- β1. Control cultures were treated with either saline or A d/luciferase. Newly synthesized proteoglycans were assessed by 35S- 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 noncollagen protein.
Results
Culture treated with A d/TGF- β1 showed 3 fold increase in proteoglycan synthesis (p<0.05), culture with exogenous TGF - β1 failed to demonstrate increase in proteoglycan synthesis. In collagen and noncollagen synthesis, cultures with A d/TGF- β1 and exogenous TGF- β1 showed similar 3.7 fold increase in collagen and 2.7 fold increase in noncollagen synthesis comparing control (p<0.05).
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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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