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IL-1beta sensitizes rat intervertebral disc cells to Fas ligand mediated apoptosis in vitro

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Abstract

Aim: To determine the apoptotic effect of recombinant rat Fas Ligand on rat inter-vertebral disc cells pre-treated with IL-1beta in vitro, and the expression of Fas in cultured rat intervertebral disc cells. Methods: Cells were isolated from the inner annulus fibrosus and transition zones of lumbar discs from Sprague-Dawley rats. The cells were grown in monolayer and divided in 5 treatment groups. IL-1beta (10 ng/mL), FasL (5, 20 ng/mL) with/without IL-1beta (10 ng/mL) pre-treatment was respectively added in Dulbecco's modified Eagle's medium and Ham's F-12 medium with 1% fetal bovine serum. After 32 h, the cells were stained with annexin V-FITC and propidium iodide to evaluate apoptosis using flow cytometry and to analysis transcription of Fas using RT-PCR. Results: Compared with control group, FasL (20 ng/mL), IL-1 (10 ng/mL)+FasL (5 ng/mL), and IL-1 (10 ng/mL) +FasL (20 ng/mL) induced significant apoptosis of the disc cells (P<0.01). Apoptosis was also induced by FasL 5 ng/mL (P<0.05); whereas, apoptosis was not induced by IL-1 (10 ng/mL) (P>0.05). IL-1 (10 ng/mL) enhanced the apoptosis-inducing effects of FasL (5 ng/mL) and FasL (20 ng/mL) in disc cells. Fas gene transcription in all groups and Fas expression in the 5 treatment groups were approximately 1.2–2.1-fold greater than control group (respectively, P<0.05). Additionally, Fas expression in FasL with IL-1 pre-treatment groups were significantly up-regulated than in FasL groups (P<0.01). Conclusion: The results of this study showed disc cells pre-treated with IL-1beta increased apoptotic rate in response to FasL in vitro and provided insights to understand Fas/FasL system-mediated apoptosis in disc cells which would be enhanced due to inflammation factor in degenerative disc.
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Keywords: Fas ligand; apoptosis; flow cytometry; interleukin-1beta; intervertebral disc cell

Document Type: Research Article

Affiliations: 1: Department of Orthopaedic Surgery, the Second Affiliated Hospital of Sun Yat-Sen University, Guangzhou 510120, China 2: Department of Lin Bai-Xin Medical Research Center, the Second Affiliated Hospital of Sun Yat-Sen University, Guangzhou 510120, China

Publication date: 2007-10-01

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