아데노바이러스를 이용한 IGF-1, TGF-β1 유전자 전달에 대한 인간 추간판 세포의 기질 생성 반응

Abstract

Background : Genetic modification of cells through gene transfer gains popularity as a sophisticated delivery system in the management of musculoskeletal diseas e. Anabolic growth factors like IGF-1 BMP-2 OP-1, and TGF-β1 were candidate for therapeutic purposus regenerating matrix of intervertebral disc. TGF-β1, 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 sign al transduction pathway to upregulate matrix synthesis. Purpose : To elucidate the anabolic effect of the combination gene transfer(IGF-1 and TGF-β1 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-TGF-β1 construct(Ad/TGF-β1) and Ad/IGF-1 respectively, second, with combination of two viral constructs(AD/TGF-β1+Ad/IGF-1). Cultures treated with saline and Ad/luciferase served as control. Then cultures were incorporated into alginate beads. Exogenous TGF-β1(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 w ere statistically significant 2.9 fold(Ad/TGF-β1) 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-β 1+Ad/IGF-1) increase in newly synthesized proteoglycan comparing control(p<0.05). Culture treated with TGF-β 1(2ng/ml) showed 3.9 fold increase and IGF-1(100 ng/ml) 2.9 fold increase, TGF-β1+IGF-1 4.3 fold increase in proteoglycan synthesis compared to control. Conclusion : Combination gene therapy provided efficient mechanism of upregulating matrix regeneration of the human intervertebral disc cells.