퇴행성 추간반과 비퇴행성 추간반에서 Cytokine 을 이용한 세포치료에 따른 추간반 재생능의 차이

Dept. of Medicine/박사There are at least four different classes of molecules that are currently being investigated for disc therapy: anti-catabolics, mitogens, morphogens, and intracellular regulators. The result of biological therapy can be also different according to therapeutic modalities and to the degree of degeneration which has a relation to chemical composition and histologic changes of intervertebral disc (IVD). The objective of this study was to investigate the molecular biologic responses of various genes and proteins relating disc degeneration to cytokines that influence disc-cell metabolism and phenotype. The responsiveness according to the degree of disc degeneration in living human IVD to these cytokines was also evaluated.Living human disc specimens were obtained from 12 patients who underwent discectomy. Disc degeneration was graded on routine T2-weighted MRI using the Pfirrmann’s grading system. The disc specimens were classified into two Groups. Group 1 (6 patients) was mild degeneration of IVD and Group 2 (6 patients) was severe degeneration of IVD. Intervertebral disc materials were taken from the patients during the discectomy. Each disc cells (2 x 105 cells/well) were grown as monolayer cultures for 6 days. After 6 days, mRNA expression of aggrecan, type I collagen, type II collagen, Sox9, alkaline phosphatase, osteocalcin, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were constructed using the complete mRNA. The gene expression was analyzed after treatment using four cytokines. Recombinant human bone morphogenic protein-2 (rhBMP-2) and transforming growth factor-β (TGF-β) were used as morphogens for the disc cells. Interleukin-1β (IL-1β) and Tumor necrosis factor-α (TNF-α) were treated as inflammatory mediators implicated in disc degeneration. The mRNA expression from disc cell culture without cytokines was used as a baseline control value. The mRNA expression of Group I was compared to Group II and reported as a ratio. The immunoreactivity of IVD for rhBMP-2 and TGF- β were analyzed to check the chondrogenic activity.Four cytokines, including rhBMP-2, TGF-β, TNF-α, and IL-1β were used as morphogenic cytokines and inflammatory implicators in this study. The responsiveness to these four cytokines between Group 1 (mild degenerative IVD) and Group 2 (severe degenerative IVD) were checked into gene and protein expression and showed statistical difference. The mRNA gene expression in Group 1 was significantly greater for aggrecan, type I collagen, type II collagen, alkaline phosphatase, osteocalcin, and Sox9 than the mRNA gene expression in Group 2 when they were not treated with cytokine. The mRNA levels of gene for these molecules after treatment of morphogens also revealed significant increment in both Groups which were much higher in Group 1 than in Group 2. There was no statistical significance in both Groups after treatment of inflammatory implicators. Micrographic findings of rhBMP-2 and TGF-β immunoreactive IVD cells for aggrecan, alkakine phosphatase, type I collagen, type II collagen, osteocalcin, and Sox9 revealed similar results in both Groups. The average numbers of immunofluorescence positive stained IVD cells for alkaline phosphatase were increased after treatment of rhBMP-2 and TGF-β in Group 1. The treatment of rhBMP-2 and TGF-β increased the expression of the various genes associated with matrix synthesis, including aggrecan, alkaline phosphatase, type I collagen, type II collagens, osteocalcin, and Sox9. The treatment of TNF-α and IL-1β decreased the expression of these genes. The molecular biologic responsiveness to the treatment of rhBMP-2, TGF-β, TNF-α and IL-1β in the degenerative living human IVD can be different according to the degree of degeneration of IVD.ope