| Background and objectiveDegenerative disc disease (DDD),degeneration of the intervertebral disc (IVD) is an underlying etiology in many of these disorders and is known to be a significant source of patient pain and morbidity. It can manifest clinically as disc herniation, radiculopathy, spinal stenosis, instability and low back pain. Despite the effect of degenerative conditions of the spine , relatively little is known about the specifics of pathophysiology compared with other disease processes. In humans, the large, round nucleus pulposus cells, thought to be of notochordal origin, disappear rin early life (childhood) leaving a scant, chondrocyte-like population of cells to maintain the disc matrix. Epidemiologic studies have implicated both hereditary and environmental factors in the genesis of disc degeneration.While risk factors for disc degenerationare well known, relatively little is known regardingthe cellular and molecular events that accompany discdegeneratin.One reason for the scarcity of disc gene expressiondata is the technical difficulty of performing gene expression analysis in disc tissue. Both the annulus fibrosus and nucleus pulposus are composed of a sparse, metabolically quiescent cell population and abundant extracellular A powerful tool to investigate the changes within the IVD during degeneration is to measure the levels of genes thought to play important roles in pathogenesis. Previous attempts to measure gene expression in IVD cells and tissue have used qualitative or semiquantitative polymerase chain reaction methods, but these techniques are highly subjective and the sensitivity is very low Quantitative real-time PCR allows a highly sensitive quantification of transcriptional levels of the gene of interest in a few hourswith minimal handling of the samples.Along with swift development of molecular biology, 1990s saw the born of DNA chip , a landmark of biological technique . Affymetrix Corporation in American took the lead place in researching in this field. In 1992, this corporation exerted the technique of to compose oligonucliotides in situ on the glass chip of around 1cm~2, and made a success in developing the first gene chip in the world . The techniques , such as fluorescent marking of probe , laser co-focused scanning and computer analysis , made progresses at the same age . In 1995 , the first microarray , which consisted of cDNA probes , was developed in Stanford University in American . These symbolized that the technique of microarray had entered in an age of wide research and application . The principle of intensive and parallel dispose of information in technique of microarray , enables it to possess incomparable characteristics, which are great efficiency, rapidness, and high-throughour analysis . The appearance and development of microarray played important role in the research of gene polymorphism , relativity between disease and gene , and exploitation of genic drugs.Microarray is the technique that a large number of genes are arranged orderly on the carrier , such as glass chip or something in high density . Data are obtained by examining the signal of fluorescence , and analyzed and compared by computer software . A great lot of genes can be examined rapidly, accurately, and effectively in one esperiment . The occurrence and evolution of DDD is related to abnormity of various genes' functions in cells . In order to find out the alteration of genes through the entire course of it, we shoule focus on the dynamic changes of genome in various stages from normal to degenneration , more than on investigation of a few genes . Using conventional methods, we can but proceed with single gene of single protein to investigate the relationship between disease and gene functiong . Since not only great deal of manpower , material resources , and financial are consumed , but also the niformation obtained is frequently simple , it is quite difficult to understand themechanism of occurrence and evolution of the disease comprehensively . We shall discover plenty of genes , which express differently in DDD , by investigating gene espression profiles using technique of microarray . More over , analysis of the functions of these differently expressed genes can promote the research on molecular biology of DDD,and help us to discover more gene markers or protein markers of DDD . This is very significant to prophylaxis , diagnosis , and treatment of Degenerative disc disease (DDD) .Methods1. Screening of the DDD related genes with cDNA microarrayThe PCR products 5069 human genes were spotted onto a kind of chemical-material-coated-glass slides.The total RNAs were isolated from the tissues.The mRNAs from both degenerated and normal lumbar intervertebral disc in humans were reversely transcribed with fluorescent dUTP.The mixed probes were then hybridized to the CDNA microarray was scanned for the fluorescent signals and analyzed by computer image analysis.2. Identification of the DDD related genes with real — time fluorescent quantitative PCR.Quantitative real-time PCR allows a highly sensitive quantification of transcriptional levels of the gene of interest in a few hourswith minimal handling of the samples. ResultsHistology confirmed progressive degeneration of the discs. Different patterns of gene expression were observed in control and degenerated discs. The degeneratede discs caused a marked upregulation of the expression of CD36, FGF2 ,VEGFB , FN, MMP-1, MMP-3, MMP-13, BAX,and Fas genes,whereas that of BCL2, TIMP1, and PTK2, Col II genes snd the expression of the decorin c was downregulated... |
PDF Full Text Request