Project description:The transcriptional events accompanying synaptogenesis are largely unknown, or have been studied in systems in which synapse formation occurs gradually over time. With a system in which synaptogenesis is synchronized and controllable, molecular or biochemical techniques can be used to examine cellular events across cultures on a wide scale, as synapses develop. Here, we have triggered synaptogenesis in immunopurified subplate neurons by coculturing with cortical feeder layers and have used microarrays to investigate the transcriptional events occuring in this more defined and controllable system. Experiment Overall Design: Affymetrix rat GeneChip microarrays were used to assess whether coculturing induces transcriptional changes in subplate neurons. Subplate synapses develop rapidly following coculturing; an exposure to the feeder layer of only 48 hours is sufficient to detect a significant increase in the density of synapses, and is as effective as longer durations. Within the first few minutes or hours of a cell's response to an exogenous signal, any primary transcriptional events are expected to involve activation of immediate early genes (IEGs), often transcription factors themselves, which may then turn on downstream target response genes encoding factors to be delivered to sites of action, such as synapses. With the aim of searching for such downstream genes, microarrays were performed after 24 hours of coculturing, an intermediate timepoint between the likely phase of IEG transcription and the large wave of synaptogenesis seen between 24 and 48 hours of coculturing. Additional microarrays were also performed after 96 hours of coculturing to assess transcriptional changes that might occur over longer timescales. Five biological replicates of control and cocultured neurons at 24 and 96 hours were used, for a total of 20 microarrays.
Project description:Inflammation is a key component of pathological angiogenesis. Here we induce cornea neovascularisation using sutures placed into the cornea, and sutures are removed to induce a regression phase. We used whole transcriptome microarray to monitor gene expression profies of several genes
Project description:Knee osteoarthritis (KOA), as a degenerative multifactorial disease, affects the quality of life and mental health of patients, and also brings a huge socioeconomic burden. Treating synovitis have shown promise as anti-inflammatory therapeutics in mitigating OA symptoms and disease progression. Here, by analysing synovial single-cell sequencing (scRNA-seq) data from KOA, we found that synovial fibroblasts (FLS) in OA synovium showed a distinct pro-inflammatory phenotype. We collected synovial tissue from patients with clinical OA as well as from healthy donors, and histological examination was consistent with findings in scRNA-seq. Inspired by recent cross-tissue fibroblast lineage studies, we identified by sequencing that healthy FLS in synovial tissues share transcriptome-level similarities with dermal fibroblasts (DFb). Subsequently, we revealed the local as well as systemic distribution of intra-articular injected DFbs by constructing/extracting two types of rat fibroblasts (luciferase DFbs as well as GFP DFbs). The results demonstrate that DFbs can be locally retained in the synovium for up to three weeks following targeted engrafting on it. And intra-articular injection does not result in DFbs migration to vital organs or the occurrence of histological changes in these organs. A rat model of KOA was constructed by anterior cruciate ligament transection (ACLT) in order to study the therapeutic effect of DFbs on KOA. After injection, the rats showed improvement in painful gait. In addition, histological as well as imaging results showed reduced synovitis and improvement in articular cartilage. Finally we verified the protective effect of DFbs on cytokine-stimulated chondrocytes in a co-culture system.
Project description:The transcriptional events accompanying synaptogenesis are largely unknown, or have been studied in systems in which synapse formation occurs gradually over time. With a system in which synaptogenesis is synchronized and controllable, molecular or biochemical techniques can be used to examine cellular events across cultures on a wide scale, as synapses develop. Here, we have triggered synaptogenesis in immunopurified subplate neurons by coculturing with cortical feeder layers and have used microarrays to investigate the transcriptional events occuring in this more defined and controllable system. Keywords: treatment type comparison, time course
