| Background:Waist and leg pain caused by intervertebral disc herniation is a common disease, has seriously affected the people's normal life. Mechanical compression is a direct factor leading to pain, but in clinical practice, we sometimes find that the clinical manifestations in patients with discrepancies in the imaging examinations, the degree of protrusion and compression are inconsistent with clinical symptoms, nerve root was found congestion and edema, similar to the inflammation-like changes. Therefore, the only mechanical compression factors to explain the pathogenesis of low back pain is not enough, inflammatory factors in the role of disc degeneration are increasingly attracted everyone's attention. This fact also shows that, the treatment of degenerative disc disease still has many problems, needed to further research and exploration. In recent years, some people reported that they have found the inflammatory cytokines in degenerative disc, therefore speculated that intervertebral disc degeneration is closely associated with the inflammatory factors. However, the exact role of the inflammatory cytokines in the process of disc degeneration is unclear.Objective:To study the effects of interleukin-6 on matrix metalloproteinase-3 and tissue inhibitor of metalloproteinase-1 gene expression in cultured human nucleus pulposus cells in vitro,to further clarify the pathological mechanism about IL-6 mediated matrix metalloproteinases in the process of intervertebral disc degeneration, so as to provide a theoretical basis for prevention and treatment the intervertebral disc degeneration.Method:1st, separates and digests the human nucleus pulposus organization to obtain the single cell and to complete the original generation of cell vaccination raise.2nd, uses HE dyeing legal time observes the cellular form, the multiplication change under the inverted microscope, the immune cell chemistry dyeing examination the expression of collagen type II. 3rd,establishes the control group and the experimental group after the original generation of cell fusion spreadin to the third generation (infliction 10μg/L,100μg/L,1000μg/L different density interleukin-6 stimulation growth).4th, the message RNA(mRNA) expression of matrix metalloproteinase-3, tissue inhibitors of matrix metalloproteinase-1 were assayed by Reverse-Transcriptase polymerase chain reaction(RT-PCR) methods.5th, draws the conclusion after the statistical software analysis.Results:1st, continuously observated under the inversion phase contrast microscope, more than 95% cells adherented after 10 days, original generation of cell many for spindle-shaped, has the pseudo pod to stretch out; After the pancreas enzyme digestion transfer of generation, the cell pastes the wall growth speed to speed up.2nd, HE dyeing cell many for spindle-shaped, has the pseudo pod to stretch out, the cell nucleus is the circular or the ellipse, the encompassment cell nucleus, in the cytoblastema obviously many vacuoles; II The collagen immune cell chemistry dyeing presents the masculine result.3rd, Stimulate human nucleus pulposus cells in vitro with IL-6 (0,10,100, 1000ng/ml) respectively, isolate total RNA and collect culture media after 72h. The message RNA(mRNA) expression of Matrix--Metalloproteinase-3,Tissue Inhibitors of Matrix Metalloproteinase-1 were assayed by Reverse-Transcriptase polymerase chain reaction(RT-PCR) methods. After the variance analysis examination, the F values were 26.360,86.790, P values are 0.000, explained four groups of between comparison differences were significant. In different concentration of IL-6, Matrix metalloproteinase-3 gene expression is up-regulation with the dose of IL-6. In different concentration of IL-6, tissue inhibitors of matrixmetalloproteinase-1 gene expression is down-regulation with the dose of IL-6. Pairwise comparison between the four groups obtained in the control group, 10μg/L,100μg/L group,1000j.g/L comparison between the groups were statistically significant (P<0.05).Conclusion:This experiment provided simple, the effective method for culturing the intervertebral disc cell.IL-6 can promote matrix metalloproteinase-3 gene expression and inhibit tissue inhibitor of metalloproteinase-1 gene expression in cultured human nucleus pulposus cells in accordance with a dose-dependent manner.At higher concentrations, IL-6 inhibits matrix synthesis of the human nucleus pulposus cells and promotes intervertebral disc degeneration.
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