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Abstract
Background
Genetic modification of cells through gene transfer gains popularity as a sophisticated delivery system in the management of musculoskeletal disease. A nabolic growth factors like IGF- 1BMP- 2 OP- 1, and TGF- ß1 were candidate for therapeutic purposus for regenerating matrix of intervertebral disc. TGF- ß1, OP- 1 and BMP- 2 share same pathway i.e. Smad while IGF- 1 utilize P13K pathway. A ccordingly 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- ß1 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. A fter isolation and culture of the cells, cultures were transduced with first, A denovirus- TGFß1construct(A d/TGF- ß1) and A d/IGF- 1 respectively, second, with combination of two viral constructs(A d/TGF- ß1+A d/IGF- 1). Cultures treated with saline and A d/luciferase served as control. Then cultures were incorporated into alginate beads. Exogenous TGF- ß1(2 ng/ml) and IGF- 1(100 ng/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(A d/TGF- ß1) and 1.8 fold(A d/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(A d/TGF- ß1+A d/IGF- 1) increase in newly synthesized proteoglycan comparing control(p<0.05). Culture treated with TGF- ß1(2ng/ml) showed 3.9 fold increase and IGF- 1(100 ng/ml) 2.9 fold increase, TGF- ß1+IGF- 1 4.2 fold increase in proteoglycan synthesis compared to control.
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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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