| BackgroundsDisc degeneration is the root cause of spinal anatomy and dysfunction disorder. Associated with spinal disc degeneration disease (disc herniation, degenerative spinal stenosis, degenerative spondylolisthesis and spinal instability, degenerative scoliosis, etc) increasing the incidence as the rhythm of life and society to speed up the aging process. Domestic and foreign scholars have got a certain understanding about its occurrence and development which addresses a relevant factor in disc degeneration, such as matrix metalloproteinase, protein polysaccharide enzymes, elastics, inflammatory mediators, cell factor; autoimmune response, nitrogen oxide and so on, but know little about the underlying causes of degeneration. In order to study the pathophysiology of disc degeneration mechanism, to explore the root causes of the occurrence and development, We applied proteomics methods in normal and degenerative intervertebral disc for two-dimensional electrophoresis and mass spectrometry,and discovered that the degeneration of nucleus pulposus in Peroxiredoxin II (Prx II) reductase exist in significant differential expression. But the role and its mechanism is unknown.Peroxiredoxins (Prxs) is a newly discovered molecular weight (20-30)×103 KD Peroxides family members, Prxs'function is diverse, including antioxidant, regulation of intracellular signal transduction involved in cell cycle progression and play a molecular chaperone and so on. Antioxidant role is the most important function H2O2 is one of the major substrates of Prxs, cells ues Prxs remove H2O2which is harmful to cells to achieve the purpose of protecting cells from damage. Recently years, however, H2O2 as second messenger signal transduction was paid more and more attention, and thus as an important regulator of Prxs has also become a research hotspot. Although previous studies have mostly focused on the Prxs on the protection of cells involved in cell signal transduction, cell differentiation, anti-oxidation, in recent years some studies found that its function is not limited to this. This article seeks to disc nucleus through the in vitro cultured Cells with the PrxⅡ, degeneration was observed on the role of nucleus pulposus cells to investigate its role in disc degeneration.ObjectivesIn vitro cultured primary degenerative intervertebral disc nucleus pulposus cells and passaged. Nucleus pulposus cells of the second generation in transmission with different concentrations of PrxⅡ,then observe the morphological changes of nucleus pulposus cells under the Inverted microscope, to detect nucleus cell activity with cck-8 kit, detect of typeⅡcollagen synthesis with ELISA. To research the role of PrxⅡin the process of disc degeneration.MethodsThe research was dived into three parts. First:to get nucleus pulposus, disc cells in primary culture, identification and passage; Second:Experiment with mass second-generation cells, establishment of the control group and experimental group, and control group is without Prx-II, the experimental group were different concentrations of Prx-Ⅱ(lOng/mL, 100ng/mL,1000 ng/mL), in 1,3,5,7 days in each group were observed morphological changes, cck-8 cell viability kit. the establishment of the control group and experimental group, the experimental group was treated with different concentrations (10ng/mL,100 ng/mL,1000 ng/mL) Prx II, the control group was treaded with nothing.at the 1,3,5,7days observe the morphological changes of cells in each group, detect activity with cck-8 kit; and third part:at 3,7 days in the control group and experimental group obtained supernatant, using pairs of antibody sandwich enzyme-linked immunosorbent assay measured the expression of collagen type II.Results1. Continuous observation was being under an inverted microscope, the original generation of nucleus pulposus cells adherent in 6-7 days, primary cells are mostly spindle-shaped, with protruding pseudopodia; Nucleus pulposus cells were digested with trypsin and passaged, the cells adherent growth rate accelerated, reached its peak after 11 days growth, after that cell growth was slowing down. The fourth generation of cell spread to a dramatic drop in apoptosis. The growth of cells within prxⅡwas slowed, growth cycles were shorted.2. Collagen II immunohistochemical staining of the cytoplasm and nucleus showed granular yellow staining positive results, which confirmed nucleus pulposus cells, and the experimental group stained lighter than the control group.3. Intervertebral disc cells absorbance (OD) values are determinated with cck-8 kit, the OD values through the factorial analysis of variance test we can see F=213.780, P=0.000, explain the difference between the five groups were significant. The same time comparison between two different groups were statistically significant differences (P<0.05); the same between groups at different time points were significant differences (P<0.05).4. we measured 3 days and 7 days OD value of typeⅡcollagen by ELSA, calculate concentration of collagen typeⅡ, using factorial analysis of variance derived that F= 126.340, P= 0.000, which shows the experimental group between control group has significant difference; Obtained by single factor analysis of variance:the 7 days between any two different groups were statistically significant differences (P<0.05), at the 3rd day, there are differences between the Control group and experimental group, there is no significant differences between the two groups of 10ng/ml and 100ng/ml, and it may be caused by errors; The same groups were significantly different with the t test (P<0.05) at different time points. This indicates that with the passage of time, apoptosis increased. PrxⅡcan reduce cell proliferation, and in a certain range of concentration in a dose related effect.ConclusionsIn this study, the training for the intervertebral disc cells provides a simple and effective method. By adding peroxidase in vitro cultured human degenerated lumbar intervertebral disc cells, the intervertebral disc cells decreased, activity was reduced, synthesis of typeⅡcollagen reduced with the increasing concentration of PrxⅡ. The test indicated thatⅡperoxidase has significant inhibition on activity of the intervertebral disc cells,Ⅱcollagen and synthesis. We can get the speculation which PrxⅡon the inhibition of nucleus pulposus cells may lead to disc degeneration as a contributing factor. |
PDF Full Text Request