Identification Of The Extracellular Matrix Proteins Of The Intervertebral Disc By Two Dimensional Electrophoreses And Mass Spectrometry

Intervertebral disc is one kind of cartilaginous tissue which is composed of tremellose nucleus pulposus in the center and annular fibrosus arranging in concentric circle one the periphery, endure the loading of the body. It will result in many symptoms in the local and whole body when it is on the process of the degeneration. Most of the basic studies on the disc degeneration disease before concentrate on the collagen proteins including genes and protein level. A number of molecules present in the matrix regulate and direct the collagen fibril assembly by interacting with the collagen molecule and also the formed fibril. Recently studies show that matrix components may play crucial keys, the collagen fibril network`s disaggregation maybe result from the matrix components foundation losing, and this is the beginning of degeneration. We didn't found anything about the disc degeneration with the proteomics method from recent publications.For the study of degeneration disease, we want to find the biomarkers in order to early diagnose. The variation of the matrix components may prior to the collagen`s, and some binding sites of the proteins should be the foundation points of the drugs, so it is crucial to do the research work with proteomics method on the matrix components.The preliminary analysis of the normal tissue of the intervertebral disc with the method of proteomics including two-dimension electrophorosis and mass spectrometry has been done in the project.Part I Preliminary experiment: Identification of the extracellular matrix of the bovine intervertebral disc by two dimensional electrophoreses and mass spectrometry Objective: to verify the feasibility of the experiment by the method of the proteomics including protein extraction, two dimensional electrophoreses and mass spectrometry; Methods: extract the proteins from bovine intervertebral disc with the method of Heinegard, separate the proteins by the SDS-PAGE, cut the strip from the gels, digest the gels with trypsin and analyze with the mass spectrometry; Result: the proteins can be separated from 16—250Kd of molecular weight, identified by the mass spectrometry, didn't find collagen proteins from samples, so the method cant destruct the network of the collagen.Part II Identification of the extracellular matrix of human normal intervertebral disc by two dimensional electrophoreses and mass spectrometryObject: to identified extracellular matrix proteins of human normal intervertebral disc by the methods of the preliminary experiment; Method: extract the proteins with methods of Heinegard,...