Identification Of The Proteins Of The Normal And Degenerative Intervertebral Disc By Two-dimensional Electrophoresis And Mass Spectrometry

Lumbar spine is the hub of body weight and activity, physiologically lordosis, the posterior intervertebral disc is thin but the anterior one is thick, the water content of the fibrous ring and nucleus gradually reduce with age, the protein mucopolysaccharide also declines year by year, the collagen fibers are gradually dissolved, the nucleus would lose the elastic and expanding properties, thus the disc is prone to shrinkage, less flexible and other degenerative diseases resulting from external force, the nucleus may also break outside through the weak part of fibrous ring.It shows that the factors of lumbar disc degeneration are diverse and complex with the mechanism research of lumbar disc degeneration deeper and deeper. Mechanics leading to the lumbar disc breaking outside after structural changes is a relatively specific pathological process, there are many complex factors in the process of structural change at lumbar intervertebral disc, such as chemistry factors and biological factors, the former study shows that degenerative lumbar disc spontaneously produce nitric oxide, interleukin-6 and prostaglandin E2, nitric oxide as a neurotransmitter plays a vasodilative role in inflammation and inhibits the synthesis of IL-6 and proteoglycan synthesis, and IL-6 promotes the expression of matrix metalloproteinase inhibitor-1.The latter study focuses on proteomics research, but it focuses largely on the changes of collagen, elastin and proteoglycan,etc.in recent years, while the overall protein research reports between the normal and degenerative disc are rare.In order to reveal the overall different proteins and their mechanism between the normal and degenerative disc in-depth as well as to provide the biological treatment of degenerative disc disease a new way, we have adopted a proteomics approach, including two-dimensional electrophoresis and matrix assisted laser adsorption/ionization time-of flight mass spectrometry (MALDI-TOF-MS), to carry out preliminary studies of different protein between the normal and degenerative disc more systematically and obtain better results.Part I : The two-dimensional electrophoresis of normal anddegenerative lumbar intervertebral disc proteinsObjective:To establish two-dimensional electrophoresis of normal and degenerative lumbardisc protein and explore the differences between them.Methods:(1) Six normal and six degenerative lumbar intervertebral disc organization were provided by volunteers, between 21-45 years old, 6 men and 6 women. Based on the range of L3～L5.(2) To remove the aggrecan by cesium chloride gradient centrifugation, proteins was separated by two-dimensional electrophoresis and identified by matrix assisted laser adsorption/ionization time-of flight mass spectrometry(MALDI-TOF-MS).Results: The electrophoresis between Normal lumbar disc and degenerative lumbar disc exist significant differences, these proteins can be well separated within pH 3～10 and molecular weight 16,000～250,000; the electrophoresis patterns of normal lumbar intervertebral disc protein presents uniform distribution status, no significantly different protein point; while the electrophoresis patterns of degenerative lumbar intervertebral disc protein shows obviously different distribution status, wecan see some different protein points.Conclusions:The most of proteins in normal lumbar intervertebral disc tissue are collagen, itis the root causes of normal disc electrophoresis patterns showing uniform status.The collagen of degenerative lumbar intervertebral disc is hydrolyzedly damaged and significantly reduced, while other proteins increase, both of them lead to a significantly different distribution, which is the root of MS analysis.Part II: MALDI-TOF-MS of different proteins between normal anddegenerative lumar intervertebral disc.Objective:To identify differentially expressed proteins between normal and degenerativelumbar disc and clear the nature of these proteins.Methods:To select 16 significantly different protein points from the results oftwo-dimensional electrophoresis between normal and degenerative lumbar disc, to identify these protein points by matrix-assisted laser desorption flight time mass spectrometer.Results:We have identified 16 significantly different protein points according to test results matching with the peptide mass fingerprint of proteins existing in the database after mass spectrometry, then we found six kinds of protein which were statistically significant based on Protein Score values in GPMAW 8.00 DEMO.Conclusions:Normal and degenerative lumbar intervertebral disc proteome exist significant differences, these different proteins property can be effectively identified by mass spectrometry; 16 different proteins in two-dimensional electrophoresis which do not have statistical significance consider that they are the collagen composition or decomposition of protein fragments, and these fragments can not be searched out with statistically significant results in the database.Conclusion:1. Collagen-II occupies the vast majority of proteins and small molecule proteins are significantly less in the normal lumbar intervertebral disc tissue. Protein types and content change markedly in the process of degeneration at lumbar intervertebral disc tissue,collagen is largely destroyed and significantly reduced, while other types of protein increase at the same time, even generate new types of protein which do not have in the normal lumbar intervertebral disc tissue.2. Different proteins can be effectively isolated from normal and degenerative lumbar disc tissue through two-dimensional electrophoresis which bases on the principle of the charge load and the experimental molecular weight of protein, and matrix-assisted laser desorption flight time mass spectrometer can identify the nature of these different proteins, all of these lay a foundation for further clarifying the mechanism of these different proteins.3. We clearly found that degenerative lumbar disc proteomics had changed markedly through application of two-dimensional electrophoresis and mass spectrometry technology, and found some statistically significant differences in protein, buried a foreshadowing for further proteomics study of degenerative lumbar intervertebral disc, as well as provided a broader and more profound possibilities to biological prevention and treatment of degenerative disease in lumbar intervertebral disc.