Expression Of Peroxiredoxin Ⅱ In Nucleus Pulposus Of Lumbar Intervertebral Disc, And The Effects In The Process Of Degeneration Of Intervertbral Disc

Back pain is strongly associated with degeneration of the intervertebral disc. As a intact and solitary constitutional unit, lumbar intervertebral disc was composed of annular fibrosus, nucleus gelatinosus and cartilage endplate. Intervertebral disc was liability to degeneration for physiological and pathological factors. The degeneration of intervertbral disc was the mian pathological change in the spinal disorders. The degeneration of nucleus gelatinosus was more severe than that of annular fibrosus and cartilage endplate. The noticeable change of degenerative nucleus gelatinosus was the loss of PG and water, the transform of the collagen type. The loss of PG and water may directly influence the biological function of intervertebral disc (IVD), then cause a serial of clinical symptom.Much progress was obtained profit from the bioresearch of diseases of degenerative intervertebral disc (DDD), specially from the application of proteome technique. From now on, reseachs on the IDD focus on the gene genetics, biomechanics and molecular biology. Gene genetics and biotic environment may be the cause of disc degeneration. And molecular plays an important role in the process of IDD including MMPs, IL-1, NO, TGF-β, BMP etc. MMP-1 and MMP-3 which belong to MMPs family may change the procedure of disc degeneration. TIMPs is a multifunctional family that can specificlly inhibit the active of MMPs. The dynamic equilibrium of MMPs and TIMPs sustains the stabbility of extracellular matrix (ECM), so the disequilibrium of this balance induce the excessus degradation of ECM. Many cytokines may regulate the active of MMPs and TIMPs. IL-1 increases the secretion of PGE2 and PLA-2 in IVD cells. IL-6 revokes the loss of proteoglycan in cartilage matrix, and inhibits the synthesis of collogen in fibroblast. Furusawa demonstrate that nitrogen monoxidum can depress the synthesis of proteoglycan in IVD, the competitive inhibitor L-NMA composites by nitrogen monoxidum can inhibit the synthesis of nitrogen monxidum dose dependently, moreover, it can increases the synthesis of proteoglycan. Conversely, accelerator synthesised by nitrogen monxidum may increase the proteoglycan.Notwithstanding, reseachs on IDD are redundant and penetrating, the mechanism of IDD till unknow. Application of two-dimensional electrophoresis and mass spectrometry technology prophase, we found some statistically significant differences in protein in normal and degenerative discs tissue, peroxiredoxin 2 is one of them. From the literature retrieval, we found that Peroxiredoxin (Prx) is a family of peroxidases are present in organisms from all kingdoms, they function as the oxygen free radical elimination, signal transduction, cell multiplication etc. There are close correlation between the IDD and the function above, so we suppose that PrxⅡmay play an important role in the occurrence and development of IDD. From now on, there is few reseach on the total protein of the IDD. So we approach the expression of PrxⅡin normal and degenerative disc tissue by Western blot test, then investigate the function of PrxⅡin the occurence and process of intervertbral disc degeneration, suppling a foundation for further study on the mechaniam of degenerative intervertebral disc.Part I:The expression of Prx II in normal and degenerative lumbar intervertebral discObjective:To identify the differential expression of Prx II in normal and degeneretive intervertebral disc by Western blot.Methods:(1)42 degenerative nucleus samples were obtained from patients in the department of orthopaedics, Zhujiang hospital from 2008.3-2009.4. Samples collection was performed according to the guidelines approved by the Ethics Committee of Zhujiang hospital. All patients were diagnosed with MRI and biopsy after operation, and with conspicuous low back pain more than 1 year, including degenerative nuclear extrusion (n=16) and degenerative nuclear protrusion (n=26). There were 22 male and 20 female, with a median age of 45.5 years old (range:28-65 years old). In addition,12 fresh samples were collected from patients with fresh fracture of lumbar vertebra or Body specimens as healthy control. There were 8 male and 4 female, with a median age of 27.5 years old (range:21-56). All nucleus samples were washed immediately with sterile physiological saline (0.9%) and preserved in refrigerator at -80℃.(2)Western blot were used to investigate the expression of PrxⅡin 12 cases of nomal disc tissue and 42 cases of degenerative disc. All data was expressed as mean±standard deviation (x±s). SPSS version 13.0 was used to performed statistical analysis, and the differences in data between group were analysis using ONE-way ANOVA and Dunnett's T3 test.P<0.05 was considered statistically significant. Results:When performed Western blot to detect all the samples, we observed a band of 22kD protein among the bands of each sample. The ratios of gray scale value of purpose bands to internal standards among all cases were evaluated and analyzed. We found that there was significant difference between the normal and degenerative samples (P<0.001). Howere, there was no significant difference between the protrusions and extrusions samples(P=0.243).Conclusion:Our findings suggested that PrxⅡexpression in degenerative tissue was more plentiful than that in normal tissues, the expression of PrxⅡwasn't correlated with the degree of degeneration. So we suppose that PrxⅡeffects mianly in the initial stage of degeneration but not in the advanced stage.PartⅡ:The function of PrxⅡin the process of IDD in animal model.Objective:To establish the rabbit model of IDD, we explore function the PrxⅡeffects during the degeneration of disc with the PrxⅡinterfere.Methods:24 New Zealand adolescent white rabbits were randomly distributed into 3 treatment groups (high saturation group, low saturation group and control group) with 8 in each. To establish rabitt model of IDD by anular puncture, four weeks later,25μl 1mg/ml,10μg/ml PrxⅡwere microinjected into the high saturation group and low saturation group, respectively.1%PBS was microinjected into the control group.2,4, 12,16 weeks after the injection,2 rabbits were randomly selected in each group for MRI scan and the detect of proteoglycan and collagen. SPSS version 13.0 was used to performed statistical analysis, and the differences in data between groups were analysis using two-way ANOVA,ONE-way ANOVA, Bonferroni, Dunnett's T3 test, Kruskal-Wallis H and Mann-Whiteney test. P<0.05 was considered statistically significant.Results:Accompany by the degree of disc degeneration, the protroglycan and collagen contents decreased accordingly, the T2 which stands for the water content descents gradually, the cell population reduced in the pathological section, and were substituted by fibrocartilage.1.MRI scan results:The T2 weighting signal of IVD decreased gradually at 2,4,12,16 weeks afer the injection,the areas of nucleus pulposus diminuted gradually and vanished in the end, accompanied by the heigh decreased.The T2 scores of high saturation group was statistically lower than that of low saturation group and control group in 4th,12th and 16th weeks(P<0.05),yet there was no significant difference between the two posterior groups in the whole process(P>0.05).2.Content of proteoglycan:The tendency of proteoglycan contents in nuclues pulposus decline.The content of proteoglycan in high saturation group (75.75±30.08) were lowest,and in control group (87.88±29.18) were highest,there was significant difference between them in 4th,12th and 16th after injection. The difference between low saturation group and control group were significant different only in 16th (P=0.02).3.The content of type-Ⅱcollagen:The Immunohistochemistry show that the type-Ⅱcollagen in normal nucleus pulposus were positive staining,the staining was taper with the time, a statistically significant was observed in all the four times between the low saturation group and high saturation group(P<0.001).There were statistically different(P< 0.001) to the low saturation group(122.51±52.02) and control group(127.22±53.59)in 4th after injection. 4. Pathological results:The nucleus pulposus present as transparent jel with few fibrous tissue. The weigh of nuclues pulposus decreased gradually by the time, much pannus were observed in the 16 weeks.The change in high saturation group is most obviously.Conclusion:The procee of disc degeneration is aggravated after disc been injured, Prx II accelerates degeneration of lumbar disc dose dependently, it effects obviously at a high saturation (1mg/ml).