The Effect Of Tensile Stress On Matrix Proteins Of Intervertebral Disc Cells And Research Into Related GeneChip

Background. Degeneration of the intervertebral disc (IVD) is the main pathophysiological process implicated in low back pain and is a prerequisite to disc herniation and vertebral canal stenosis. Investigations reveal that degenerative changes of IVD are obvious in both manual workers carrying heavy things and sedentary workers whose jobs require prolonged sitting. Machine operators, drivers and porters are at high risk of low back pain and disc herniation, with the chance of heniation three times greater than ordinary adults. Such observations suggest a significant role of mechanics in progression of IVD degeneration. Degenerative changes on the biochemical level are noted first in the nucleus pulposus(NP), with a loss of glysosaminoglycans (GAG) and a change in the ratio of Collagen-I and Collagen-Ⅱ; these changes are accompanied or initiated by increased synthesis and activation of matrix degrading enzymes such as matrix metalloproteinases (MMPs). Changes in the annulus fibrosus (AF) are less evident, but are characterized by changes in collagen matrix composition and organization.Objective. To investigate the effect of tensile stress on gene expression of human intervertebral disc cells and the role of mechanics in the dynamic process of intervertebral disc degeneration. To research into related GeneChip to find out other genes affected by tensile stress so as to provide clues for advanced research.Materials and methods. IVDs were obtained from patients with disc herniation undergoing discectomy. The discs were divided into AF and NP tissues, and cells were cultured in vitro.(1) Phenotype of AF cells and NP cells were verified.(2) AF cells and NP cells were seeded in elastic membranes, and tensile stress was applied with2%, or5%, or10%elongation separately for one hour.24hours later, AF cells and NP cells were harvested and changes in expression of genes known to influence IVD matrix turnover (Aggrecan, Collagen-II, MMP-3, TIMP-1) were analyzed by real-time RT-PCR.(3) AF cells applied with10%elongation stress for one hour were analyzed by Affymetrix GeneChip3’IVT Express Kit;(4) NP cells applied with10%elongation stress for one hour were harvested immediately at the end of stimulation, or12hours,24hours and48hours later separately, and kinetics of gene expression by real-time RT-PCR.Results.(1) AF cells and NP cells grew in monolayer with a polygonal structure. Collagen-Ⅱ expression was positive.(2) In AF cells, tensile stress with2%elongation had no effect on gene expression.5%and10%stress promoted gene expression of Collagen-Ⅱ, MMP-3and TIMP-1significantly (P<0.05), but no obvious change on Aggrecan expression was detected.(3) According to the results of GeneChip,10%elongation stress for one hour led to expression changes greater than2folds in more than1600genes, and changes greater than5folds in119genes in AF cells. SERPINA1, IL-8, PRG4and TNFAIP6ranked the first four places, with the ratio to control being33.8,27.1,26.9and25.3respectively. KRT19was the most significantly suppressed gene, with the expression ratio to control being0.04.(4) In NP cells,5%stress led to significant suppression of Aggrecan expression (P<0.05) and dramatic increase in Collagen-Ⅱ, MMP-3and TIMP-1expression (P<0.05).10%elongation inhibited gene expression of Aggrecan and Collagen-Ⅱ (P<0.05), while the effect on MMP-3and TIMP-1was quite the reverse.(5) In response to10%elongation, Aggrecan mRNA and Collagen-Ⅱ mRNA decreased to minimum at12hours and0hour respectively after withdrawing tensile stress, while MMP-3and TIMP-1peaked at0hour and24hours respectively in NP cells.Conclusions.(1) The effect of tensile stess on IVD cells depended on the intensity of stress. Low-intensity stress had no effect on expression of genes known to influence AF cell matrix, while moderate-to-high intensity stress resulted in degradation or remodeling.(2) Kinetics of mRNA expression following tensile stress, regulated by intensity, duration and interval of stress, represented the process of acute injury, degradation or remodeling in NP cells.(3) Besides MMPs and TIMPs, SERPINA1and GRP4may also play important roles in metabolism of IVD matrix. It was the first time that the impact of tensile stress on mRNA expression of SERPINA1, GRP4and KRT19was investigated.