Project description:Disc Degeneration is a multifactorial disease which cause severe constant chronic pain. The development of disc degeneration could involve both genetic and environmental factors, so it's important to elucidate the difference in gene expression profiles between degenerative and non-degenerative discs from elderly patients and younger patients, respectively. Affymetrix GeneChip Human Genome U133A Array was used to derive gene expression profiles and identified genes that would express at significantly different levels between degenerative and non-degenerative samples. Human intervertebral disc tissues were harvested from elderly and younger patients with degenerative disc disease and adolescent idiopathic scoliosis, respectively, for RNA extraction and hybridization on Affymetrix microarrays. One degenerative sample was compared to one non-degenerative sample in the same microarray run, and comparative analyses were performed in triplicate. Gene whose expression levels varied across the samples with a log ratio >0.5 or <-0.5 were selected as the genes related to disc degeneration.
Project description:Research on disc degeneration has been heterogeneous in their use of control discs used for comparison with diseased discs. Discs from scoliosis, cadavers and voluntary organ donors are the common controls used in intervertebral disc research. In order to find out the ideal control among these discs, the characters of scoliotic discs and discs from MRI normal voluntary organ donors controls used in disc research has been analysed using proteomics and to establish 'True Controls' that can be utilized for future Intervertebral disc (IVD) research.
Project description:The human intervertebral disc (IVD) is a complex and dynamic structure that functions to provide spinal stability, mobility and flexibility. It comprises three main compartments: 1) a water-rich central compartment called the nucleus pulposus (NP), which is enveloped by 2) the annulus fibrosus (AF) and sandwiched between 3) two cartilaginous endplates (EP) from which the IVD gains its nutrition and provides a means to get rid of metabolic waste. The IVD is constantly under extreme conditions of hypoxia, nutrition, and loading stresses. As such, it wears with ageing, and the rates of wearing may be sped up by a multitude of risk factors. Phenotypically, the AF may become weaker that a portion of it may protrude into neighboring tissues, resulting in disc bulging, or even worse, it may rupture and form a herniation Most striking of all, magnetic nuclear imaging (MRI) of the NP may become darker, with spotted high-intensity zones (HIZs), indications of dehydration and annular disruptions (Peng, et al. 2006). In a herniated or even collapsed disc, the contents of NP may leak into the neighboring spaces and external cells (such as macrophages) may infiltrate into the NP (Nakazawa, et al. 2018), causing the disc functions to be partially or fully compromised. The underlying mechanism of these involves complex interplays of cellular and molecular changes and remains poorly understood; nevertheless, the process is suggested to be triggered and driven by both environmental and genetic factors, or their combinations. In an effort to study the molecular dynamics of the ageing process of the human IVD, we profiled the transcriptomes of two groups of individuals: a young and non-degenerated group (N=2) and an aged and degenerated group (N=2). We profiled the transcriptomes of both AF and NP for each individual.
Project description:Low back pain continues to be a major public health problem worldwide. In this study, we used single-cell transcriptomic analysis to identify new specific biomarkers for nucleus pulposus (NP) and annulus fibrosis (AF) cells and to define cell populations within non-degenerating and degenerating human intervertebral discs (IVD). Freshly isolated human NP and AF cells were separately isolated from non-degenerating (nD) and degenerating (D) discs of the same individual. Isolated cells were subjected to droplet-based single-cell RNA sequencing (scRNA-Seq) using 10X Genomics platform. A total of 3134 (AFD), 3182 (AFnD), 3665 (NPD) and 3918 (NPnD) individual cells were profiled from nD and D discs respectively.
Project description:Disc Degeneration is a multifactorial disease which cause severe constant chronic pain. The development of disc degeneration could involve both genetic and environmental factors, so it's important to elucidate the difference in gene expression profiles between degenerative and non-degenerative discs from elderly patients and younger patients, respectively. Affymetrix GeneChip Human Genome U133A Array was used to derive gene expression profiles and identified genes that would express at significantly different levels between degenerative and non-degenerative samples.
Project description:We investigated the heterogeneous cell populations composing Bovine Intervertebral Discs (IVDs) through single cell RNA sequencing technologies. The assay sequenced over 14,000 cells composing 5 bovine discs from 3 unique bovine tails. Through both established and custom analysis pipelines, we characterize cell heterogeneity between populations of Nucleus Pulposus and Annulus Fibrosus cells. We further characterize populations of Endothelial, Muscle, Immune, and Notochord.
Project description:Purpose: Degenerative changes of the intervertebral disc are a leading cause of back pain and disability worldwide. However, precise mechanisms driving the initiation and progression of pathology have remained elusive. We find that adhesion G-protein coupled receptor Adgrg6 plays a critical role in maintaining postnatal intervertebral disc homeostasis. The goal of this study is to uncover early molecular changes in Adgrg6-defeicnet intervertebral discs prior to overt histopathology. Methods: Intervertebral disc mRNA profiles of 20-day-old wild type and Col2Cre; Adgrg6f/f mutant mice were generated by deep sequencing in triplicates. Cutadapt and perl scripts were used to remove the reads that contained adaptor contamination, low quality bases and undetermined bases. Then sequence quality was verified using FastQC. We used HISAT2 to map reads to the genome of Mus Musculus (GRCm38.88). The mapped reads of each sample were assembled using StringTie. Then, all transcriptomes from biological samples were merged to reconstruct a comprehensive transcriptome using perl scripts and gffcompare. After the final transcriptome was generated, StringTie and Ballgown was used to estimate the expression levels of all transcripts. StringTie was used to perform expression level for mRNAs by calculating FPKM. The differentially expressed mRNAs were selected with log2 (fold change) >1 or log2 (fold change) <-1 and with statistical significance (p value < 0.05) by R package Ballgown. Results: We found 884 differential expressed genes with statistical significance (p value < 0.05, and with a more stringent cut-off adjusted p value <0.05 and fold-change >2, we observed 42 differential expressed genes. Enriched pathways and biological processes using gene ontology (GO) terms included extracellular matrix, positive regulation of fibroblast proliferation, extracellular matrix structural constituent conferring tensile strength, regulation of tyrosine phosphorylation of STAT protein, and ion transport. We also find significantly increased expression of fibrotic collagens, induced expression of some Suppressor of Cytokine Signaling (SOCS) genes, and dysregulated expression of some components associated with ion transport system. Several of the significantly upregulated genes are associated with biomarkers or risk of lumbar disc degeneration and osteoarthritis in humans or animal models. Conclusions: Our study provides detailed analysis of intervertebral disc transcriptomes from both wild type and Adgrg6-deficient mice, with three biological replicates, prior to overt histopathology. Our transcriptomic analysis demonstrated a robust dysregulation of several important pathways and components of the intervertebral disc homeostasis, including induction of fibrotic gene expression, alteration of ion transport component, as well as changes in some chondrogenic and catabolic factors, prior to the onset of histopathology and disc degeneration. These data strongly suggest that ADGRG6 signaling is a critical factor for the maintenance of healthy gene expression profiles in the IVD.
Project description:Intervertebral Disc (IVD) degeneration leading to Low back pain (LBP) is the most common musculoskeletal disorder. Lack of knowledge on the intricate homeostatic mechanisms necessitates proteomic characterisation of a normal human IVD to understand the biological process and unravel the pathomechanisms of degenerative disc disease (DDD). In this study, we employed proteomic approach coupled with tandem mass-spectrometry to derive the comprehensive list of proteins expressed in true biologically normal control discs. This would serve as the basis for identifying the interacting molecules participating in significant biological processes and pathways disrupted during aging and degeneration.