삼차원적으로 배양된 인간의 추간판 세포에서 다중 유전자 치료

Abstract

Purpose : Genetic modification of cells through gene transfer gains popularity as a sophisticated delivery system in the management of musculoskeletal disease. However, ideal combination of anabolic growth factor genes for matrix regeneration of the disc tissue is still unclear. Accordingly, the objective of this in vitro study was to elucidate the anabolic effect of the combination gene transfer 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-TGFβ1 construct (Ad/TGF-β1), Ad/IGF-1, and Ad/BMP-2 respectively, second, with combination of two viral constructs (Ad/TGF-β1 + Ad/IGF-1, Ad/IGF-1 + Ad/BMP-2, Ad/TGF-β1 + Ad/BMP-2), and third, with combination of three viral construct (Ad/TGF-β1 + Ad/IGF-1 + Ad/BMP-2). Cultures treated with saline and Ad/luciferase served as control. Total viral concentration was adjusted to 75MOI. Then cultures were incorporated into alginate beads. Transgene expression was assessed by ELISA. Newly synthesized proteoglycan was assessed using S35 incorporation using chromatography on Sephadex G-25 in PD-10 column. Results : Concentration of growth factor increases markedly in conditioned medium, indicating transgene expression. In cultures transduced with single therapeutic gene construct, there were statistically significant 2.9 fold (Ad/TGF-β1), 1.8 fold (Ad/IGF-1), 1.9 fold (Ad/BMP-2) 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-β1 + Ad/IGF-1), 3.2 fold (Ad/IGF-1 + Ad/MBP-2), and 3.5 fold (Ad/TGF-β1 + Ad/BMP-2) increase in newly synthesized proteoglycan comparing control (p<0.05). Cultures transduced with triple combination of therapeutic gene constructs exhibited 4.7 fold increase in newly synthesized proteoglycan comparing control (p<0.05). Conclusion : Cocktail gene therapy provided efficient mechanism of upregulating matrix regeneration of the human intervertebral disc cells.