Medicine & Health

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  • (2013) Apte, Minoti; Yang, Lu; Phillips, Phoebe; Xu, Zhihong; Kaplan, Warren; Cowley, Mark
    Journal Article
    Activated pancreatic stellate cells (PSCs) are responsible for the fibrotic matrix of chronic pancreatitis and pancreatic cancer. In vitro protocols examining PSC biology have usually involved PSCs cultured on plastic, a non-physiological surface. However, PSCs cultured on physiological matrices e.g. MatrigelTM (normal basement membrane) and collagen (fibrotic pancreas), may have distinctly different behaviours compared to cells cultured on plastic. Therefore, we aimed to i) compare PSC gene expression after culture on plastic, MatrigelTM and collagen I; ii) validate the gene array data for transgelin, the most highly dysregulated gene in PSCs grown on activating versus non-activating matrices, at mRNA and protein levels; iii) examine the role of transgelin in PSC function; and iv) assess transgelin expression in human chronic pancreatitis sections. Culture of PSCs on different matrices significantly affected their gene expression pattern. 146, 619 and 432 genes respectively were differentially expressed (p < 0.001) in PSCs cultured on collagen I vs MatrigelTM, MatrigelTM vs plastic and collagen I vs plastic. The highest fold change (12.5 fold upregulation) in gene expression in cells on collagen I vs MatrigelTM, was observed for transgelin (an actin stress fibre associated protein). Transgelin was significantly increased in activated PSCs versus quiescent PSCs. Silencing transgelin expression decreased PSC proliferation and also reduced platelet derived growth factor (PDGF)-induced PSC migration. Notably, transgelin was highly expressed in chronic pancreatitis in stromal areas and peri-acinar spaces but was absent in acinar cells. These findings suggest that transgelin is a potentially useful target protein to modulate PSC function so as to ameliorate pancreatic fibrosis.

  • (2007) Chung, Sylvia; Wei, Ai-Qun; Connor, David; Webb, Graham; Molloy, Tim; Pajic, Marina; Diwan, Ashish
    Journal Article
    Study Design. Nonviral transfection of nucleus pulposus cells with a telomerase expression construct to assess the effects on cellular lifespan, function, karyotypic stability, and transformation properties. Objectives. To investigate whether telomerase gene therapy can extend the cellular lifespan while retaining functionality of nucleus pulposus cells in a safe manner. Summary of Background Data. Degeneration of the intervertebral disc is an age-related condition in which cells responsible for the maintenance and health of the disc deteriorate with age. Telomerase can extend the cellular lifespan and function of other musculoskeletal tissues, such as the heart, bones, and connective tissues. Therefore, extension of the cellular lifespan and matrix production of intervertebral disc cells may have the potential to delay the degeneration process. Methods. Ovine nucleus pulposus cells were lipofectamine transfected in vitro with a human telomerase reverse transcriptase (hTERT) expression construct. Cellular lifespan and matrix transcript levels were determined by cumulative population doublings and real-time RT-PCR, respectively. G1-cell cycle checkpoint, p53 functionality, growth of transfected cells in anchorage-independent or serum starvation conditions, and karyotypic analysis were performed. Results. Transfection was achieved successfully with 340% +/- 7% ( mean +/- SD) relative telomerase activity in hTERT- transfected cells. hTERT transfection enabled a 50% extension in mean cellular lifespan and prolonged matrix production of collagen 1 and 2 for more than 282 days. Karyotypic instability was detected but G1-cell cycle checkpoint and p53 was functionally comparable to parental cells with no growth in serum starvation or anchorage-independent conditions. Conclusions. Telomerase can extend the cellular lifespan of nucleus pulposus cells and prolong the production of extracellular matrix. Safety is still unresolved, as karyotypic instability was detected but no l