인간 추간판 세포의 기질 생성: 유전자 전달법, 외부 투입된 성장인자, 배양 기간별, 배양기법 등에 따른 기질 생성의 변화

Abstract

Study-Design: In vitro experiment to determine the matrix synthesis of intervertebral disc (IVD) cell to various biologic interventions and conditions. Objective: To elucidate biolic responses in terms of matrix synthesis of human IVD cells in vitro to various factors i.e. concentratin of adenoviral vector and exogenous growth factor, duration of incubation, and type of culture methods. Summary-of-Literature-Review: Sophisticated method to delivery of growth factors, in continuous manner, is the genetic modification of disc cells through gene transfer. Direct comparison of gene transfer and exogenous growth factor on matrix synthesis has not been reported. Materials-and-Method: IVD tissue was obtained from twenty three patients. isolation and preparation of disc cells in monolayer (2 D) and alginate beads (3 D) culture were performed. Disc cells in 2 D and 3 D were treated with either Ad/TGF-β1 or exogenous TGF-β1. Control cultures were treated with either saline or Ad/luciferase. Matrix synthesis (newly synthesized proteoglycan) was measured in various conditions (concentratin of adenoviral vector and exogenous grwoth factor, duratino of incubation, and type of culture methods). Newly synthesized proteoglycan were analyzed using chromatography on Sephadex G-25 in PD-10 columns after S35-sulfate incorporation. Results: Ad/TGF-β1 showed increase in proteoglycan synthesis (plateau at 75 MOI) in 3 D culture, (plateau at 25 MOI) in 2 D culture. In 3 D culture, Ad/TGF-β1 showed significant increase in proteoglycan syntheisis on day 1, 2, and 3 of incubation. In 2 D culture, Ad/TGF-β1 showed singificant increase in proteoglycan synthesis on day 2 of incubation with signifiant loss of anabolic effect on day 3. In 3 D culture, exogenous TGF-β1 showed increase in proteoglycan synthesis (plateau at 2 ng/㎖) while in 2 D culture, there is no synthetic response to exogenous TGF-β1. Conclusion: Therapeutic gene gransfer provided sustained and increased anabolic responses than exogenous growth factor.