The Influence Of Proin Ammatory Cytokines IL-1β On The Growth And Expression Of Nerve Growth Factor And MMP-9in Nucleus Pulposus Cells

Objective:To explore the impact of pro-inflammatory cytokines IL-1β on the growth ofnucleus pulposus cells and the influence of NGF and MMP-9protein expression ofnucleus pulposus cells.Methods:1.Withdraw and cultivate rabbit nucleus pulposus cells to observed theirmorphology, then grow the logarithmic phase of nucleus pulposus cells in96-wellplates, treating the cells with using different concentrations of IL-1β(10ng/ml,20ng/ml,40ng/ml and80ng/ml) for24hours and of explore nucleus pulposus cellsvitality through the MTT assay.2. Passage the cells in6-well plates,3×105per well when the cell fusion arrivedat80%. There are four experimental groups, control group and marked on each hole.After incubation for24hours, change the primary medium to new medium with1%fetal bovine serum and cultured for12hours and replaced with2ml new mediumcontaining10%fetal bovine serum. Adding with40ng/ml IL-1β into fourexperimental wells to incubate cells for24h,48h,72h,96h, then collected the cells toextract total protei. Cells in control group were also cultured with2ml new mediumwith10%fetal bovine serum for96h to extract protein. Quantity the expression levelof MMP-9and NGF in different time induced by the same concentration of IL-1βthrough the Western blotting methods.3. Passage the cells in6-well plates,3×105per well when the cell fusion arrivedat80%. There are four experimental groups, one control group and marked on eachhole. After incubation for24hours, change the primary medium to new medium with1%fetal bovine serum and cultured for12hours and replaced with2ml new mediumcontaining10%fetal bovine serum. Adding dirrerent consentration of IL-1β into fourexperimental wells (10,20,40and80ng/ml) to incubate cells for48hours, thencollected the cells to extract total protein. Cells in control group were cultured with the normal medium without IL-1β. Using the western blotting methods to quantity theexpression level of MMP-9and NGF under different concentration of IL-1β.Results:1. Primary cell cultures gradually adherent into the the wells after cultured for72h hours, it seems irregular, long spindle, polygonal, slow growth. After5days,adherent cells gradually increased,accounting for50-80%, most of the cells growclose to the tissue, distributed like cloned like polygonal or spindle-shaped.Non-adherent cells or tissue gradually showed vacuolization. Cell outlines andnucleus can be seen clearly, slightly flattened translucent, under250times themicroscope. After10days the number of cells surrounding tissue culture increasedand grow fast into exponential growth phase. About15days adherent cells began toappear fusion, was "stone pavement-like."2. MTT results showed that, IL-1β in the experimental concentration (10ng/ml,20ng/ml,40ng/ml,80ng/ml) had no effect on the activity of the nucleus pulposuscells, there is no significant difference between the drug-treated four groups andcontrol group.3. IL-1β can induced the rabbit nucleus pulposus cells to express the NGFprotein and the expression level increased along with the incubation time of IL-1β,there is significant different between them(P<0.05). However the expression of NGFin the four experimental group were similar and no difference between them(P>0.05).4. IL-1β can induced the rabbit nucleus pulposus cells to express the MMP-9protein and the expression level increased along with the incubation time of IL-1β,there is significant different between them (P<0.05). However the expression ofMMP-9in the four experimental group were similar and no difference betweenthem(P>0.05).Conclusion:1. Pro-inflammatory cytokines IL-1β can stimulate MMP-9and NGF expressionin the nucleus pulposus cells, otherwise, MMP-9, NGF protein was not expressed orlow expression in normal condition.2. The expression level of NGF and MMP-9in nucleus pulposus cells inducedby inflammatory cytokines IL-1β is time-dependent instead od dose-dependent. 3. Pathophysiological changes in patients with discogenic low back pain disc,inflammatory cytokines can stimulate disc cells to express NGF, resulting in the nervefibers growing into the disc inside. On the other side the disc induce the secretion ofmatrix metalloproteinases that interfere the balance between the degrade andsynthetic of disc internal matrix, accelerating lumbar disc degeneration.