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Quantitative Reverse Transcriptase Polymerase Chain Reaction (quantitative + reverse_transcriptase_polymerase_chain_reaction)
Selected AbstractsGrowth hormone regulates osteogenic marker mRNA expression in human periodontal fibroblasts and alveolar bone-derived cellsJOURNAL OF PERIODONTAL RESEARCH, Issue 4 2003H. R. Haase Background:, Growth hormone (GH) is a potent regulator of bone formation. The proposed mechanism of GH action is through the stimulation of osteogenic precursor cell proliferation and, following clonal expansion of these cells, promotion of differentiation along the osteogenic lineage. Objectives:, We tested this hypothesis by studying the effects of GH on primary cell populations of human periodontal ligament cells (PLC) and alveolar bone cells (ABC), which contain a spectrum of osteogenic precursors. Methods:, The cell populations were assessed for mineralization potential after long-term culture in media containing ,-glycerophosphate and ascorbic acid, by the demonstration of mineral deposition by Von Kossa staining. The proliferative response of the cells to GH was determined over a 48-h period using a crystal violet dye-binding assay. The profile of the cells in terms of osteogenic marker expression was established using quantitative reverse transcriptase polymerase chain reaction (RT-PCR) for alkaline phosphatase (ALP), osteopontin, osteocalcin, bone sialoprotein (BSP), as well as the bone morphogenetic proteins BMP-2, BMP-4 and BMP-7. Results:, As expected, a variety of responses were observed ranging from no mineralization in the PLC populations to dense mineralized deposition observed in one GH-treated ABC population. Over a 48-h period GH was found to be non-mitogenic for all cell populations. Quantitative reverse transcriptase polymerase chain reaction (RT-PCR) BSP mRNA expression correlated well with mineralizing potential of the cells. The change in the mRNA expression of the osteogenic markers was determined following GH treatment of the cells over a 48-h period. GH caused an increase in ALP in most cell populations, and also in BMP expression in some cell populations. However a decrease in BSP, osteocalcin and osteopontin expression in the more highly differentiated cell populations was observed in response to GH. Conclusion:, The response of the cells indicates that while long-term treatment with GH may promote mineralization, short-term treatment does not promote proliferation of osteoblast precursors nor induce expression of late osteogenic markers. [source] Effect of porto-systemic shunting on NOS expression after extended hepatectomy in ratsHEPATOLOGY RESEARCH, Issue 1 2009Hironori Hayashi Aim:, Several surgical procedures have been developed for reducing portal vein pressure to prevent postoperative liver injury. Nitric oxide synthase expression (NOS) induced by elevation of portal vein pressure is thought to play an important role in liver regeneration, but the details are not well understood. Methods:, Rats in the control group and in the subcutaneous splenic transposition (SST) group underwent 90% partial hepatectomy. Survival and portal vein pressure were analyzed. The serum IL-6 and TNF-, levels were measured by enzyme-linked immunosorbent assay (ELISA). Hepatocyte proliferation and apoptosis 12 hours after hepatectomy were analyzed immunohistochemically. The protein and messenger RNA expression of inducible and endothelial NOS were analyzed using Western blotting and quantitative reverse transcriptase polymerase chain reaction, respectively. Results:, The survival rate of the SST group was significantly higher. Portal vein pressure, TNF-, level and the apoptotic index were significantly lower in the SST group. Twelve hours after surgery, liver inducible NOS (iNOS) protein expression was significantly lower in the SST group. However, protein expression of endothelial NOS was not significantly different between the groups. Conclusion:, Inducible NOS expression after extended hepatectomy is related to the effects of porto-systemic shunting on the splanchnic circulation. Also, iNOS induction and concomitant nitric oxide generation appear to participate in the cytotoxicity of excessive portal pressure after extended hepatectomy. [source] Molecular diagnosis of basal cell carcinoma and other basaloid cell neoplasms of the skin by the quantification of Gli1 transcript levelsJOURNAL OF CUTANEOUS PATHOLOGY, Issue 2 2005Naohito Hatta Background:, Distinguishing basal cell carcinoma (BCC) from other benign and malignant skin tumors is sometimes a difficult task for the pathologists. Because the activation of hedgehog signals and the up-regulation of its critical transcriptional factor Gli1 are well documented in BCC, a molecular technique measuring Gli1 transcripts may aide the diagnosis. Methods:,Gli1 transcript levels were measured by real-time quantitative reverse transcriptase polymerase chain reaction (RT-PCR) using RNA extracted from formalin-fixed, paraffin-embedded tissues of 68 cases of various skin tumors. Hematoxylin and eosin-stained pathology slides were independently reviewed by three expert dermatopathologists. Results:, The histological diagnoses were unambiguous in 53 tumors. The tumors included BCC (21), squamous cell carcinoma (13), seborrheic keratoses (8), trichoepithelioma (5), eccrine poroma/porocarcinoma (4), and sebaceous epithelioma/carcinoma (2). In these unambiguous cases, all BCC and trichoepithelioma tumors showed high expression of Gli1mRNA, while the expression was virtually absent in other tumors. The diagnosis was discordant among three pathologists in the remaining 15 tumors. Histological diagnoses included BCC, BCC with sebaceous differentiation, sebaceoma/sebaceous epithelioma, trichoblastoma, trichoepithelioma, basaloid follicular harmartoma, basosquamous carcinoma, etc. Six of them showed high Gli1 transcript levels. Conclusions:, Quantification of Gli1 transcripts by RT-PCR is helpful in discriminating BCC and trichoepithelioma from other skin tumors. [source] Growth hormone regulates osteogenic marker mRNA expression in human periodontal fibroblasts and alveolar bone-derived cellsJOURNAL OF PERIODONTAL RESEARCH, Issue 4 2003H. R. Haase Background:, Growth hormone (GH) is a potent regulator of bone formation. The proposed mechanism of GH action is through the stimulation of osteogenic precursor cell proliferation and, following clonal expansion of these cells, promotion of differentiation along the osteogenic lineage. Objectives:, We tested this hypothesis by studying the effects of GH on primary cell populations of human periodontal ligament cells (PLC) and alveolar bone cells (ABC), which contain a spectrum of osteogenic precursors. Methods:, The cell populations were assessed for mineralization potential after long-term culture in media containing ,-glycerophosphate and ascorbic acid, by the demonstration of mineral deposition by Von Kossa staining. The proliferative response of the cells to GH was determined over a 48-h period using a crystal violet dye-binding assay. The profile of the cells in terms of osteogenic marker expression was established using quantitative reverse transcriptase polymerase chain reaction (RT-PCR) for alkaline phosphatase (ALP), osteopontin, osteocalcin, bone sialoprotein (BSP), as well as the bone morphogenetic proteins BMP-2, BMP-4 and BMP-7. Results:, As expected, a variety of responses were observed ranging from no mineralization in the PLC populations to dense mineralized deposition observed in one GH-treated ABC population. Over a 48-h period GH was found to be non-mitogenic for all cell populations. Quantitative reverse transcriptase polymerase chain reaction (RT-PCR) BSP mRNA expression correlated well with mineralizing potential of the cells. The change in the mRNA expression of the osteogenic markers was determined following GH treatment of the cells over a 48-h period. GH caused an increase in ALP in most cell populations, and also in BMP expression in some cell populations. However a decrease in BSP, osteocalcin and osteopontin expression in the more highly differentiated cell populations was observed in response to GH. Conclusion:, The response of the cells indicates that while long-term treatment with GH may promote mineralization, short-term treatment does not promote proliferation of osteoblast precursors nor induce expression of late osteogenic markers. [source] Expression of osteopontin in chronic rhinosinusitis with and without nasal polypsALLERGY, Issue 1 2009X. Lu Background:, Osteopontin (OPN) is a multifunctional 34-kDa extracellular matrix protein that can influence the inflammatory process. However, the presence of OPN in human sinonasal mucosa and its roles in the inflammatory process of chronic rhinosinusitis (CRS) are not clear. This study investigated the expression of OPN in human sinonasal mucosa, its cytokine-driven expression regulation, and its effect on cytokine production in sinonasal mucosa. Methods:, Surgical samples were investigated by means of quantitative reverse transcriptase polymerase chain reaction for evaluation of OPN messenger RNA (mRNA) expression, and the presence and location of OPN protein expression were analyzed using immunohistochemistry. Furthermore, nasal explant culture was used to investigate the mutual regulatory interactions between interferon (IFN)-,, interleukin (IL)-4, IL-5, IL-13, IL-1,, and tumor necrosis factor (TNF)-, and OPN in sinonasal mucosa. Results:, Osteopontin expression was significantly upregulated in CRS tissues compared with control tissues. There was a further significant increase of OPN expression in patients with nasal polyps (NPs) and asthma. Immunohistochemistry revealed positive staining of OPN in epithelial cells, submucosal glands, infiltrating cells, and extracellular matrix. Osteopontin mRNA was induced by IFN-,, IL-1,, and TNF-,, but inhibited by IL-4 and IL-13. On the contrary, OPN induced IFN-,, IL-4, IL-5, IL-13, IL-1,, and TNF-, production in sinonasal mucosa. Conclusions:, The expression of OPN is upregulated in CRS. The mutual regulatory interactions between OPN and inflammatory cytokines suggest that OPN may play an important role in the pathogenesis of CRS. [source] Pressure simulation of orthodontic force in osteoblasts: a pilot studyORTHODONTICS & CRANIOFACIAL RESEARCH, Issue 1 2004U. Baumert Structured Abstract Authors , Baumert U, Golan I, Becker B, Hrala BP, Redlich M, Roos HA, Reichenberg E, Palmon A, Müßig D Objectives , To elucidate the RUNX2 gene expression induction in human osteoblasts after mechanical loading. Design , Using a stringent pulse-chase protocol human osteoblasts were exposed to centrifugal pressure force for 30 and 90 min. Untreated control cells were processed in parallel. Before, and at defined times after centrifugation, total RNA was isolated. RUNX2 gene expression was measured using real-time quantitative reverse transcriptase polymerase chain reaction. The stress/control ratio was used to illustrate possible stimulatory or diminishing effects of force application. Results , Immediately after 30 min of force application the RUNX2 gene expression was induced by a factor of 1.7 ± 0.14 as compared with the negative control. This induction decreased rapidly and reached its pre-load levels within 30 min. Longer force applications (up to 90 min) did not change the RUNX2 gene expression. Conclusion , In mature osteoblasts centrifugal pressure force stimulates RUNX2 gene expression within a narrow time frame: loading of mature cells results in a temporary increase of RUNX2 expression and a fast downregulation back to its pre-load expression level. With this pilot study the gene expression behavior after mechanical stimuli could be determined with a simple laboratory setup. [source] Prostaglandin E2 is activated by airway injury and regulates fibroblast cytoskeletal dynamics,THE LARYNGOSCOPE, Issue 7 2009Vlad C. Sandulache MD Abstract Objectives/Hypothesis: To characterize the activation of cyclooxygenase (COX)-2/prostaglandin (PG) E2 signaling during airway mucosal repair and its subsequent role during the wound healing process. Study Design: Prospective animal study. Methods: The subglottis was approached via cricothyroidotomy. Sham airways were closed, and wounded airways were subjected to laser injury and closed. Subglottic tissue was harvested at 12 hours, 24 hours, 48 hours, and 72 hours postinjury. Secretions were collected preoperatively and at time of sacrifice. Inflammatory gene expression was analyzed using quantitative reverse transcriptase polymerase chain reaction. Subglottic/tracheal explants were exposed to exogenous IL-1, in the presence or absence of COX inhibitors. Explant-produced PGE2 levels were assayed using enzyme linked immunoassays. Human airway fibroblast migration and collagen contraction were assayed in the presence or absence of prostaglandin E2. Results: Laser injury triggers a rapid, dose-dependent increase in mucosal IL-1, and COX-2 gene expression, with an anatomical distribution proportional to the distance from the site of injury. Gene upregulation correlates with dose-dependent increases in PGE2 mucosal secretion levels. Ex vivo analysis indicates IL-1, is responsible for the activation of the COX-2 / PGE2 pathway. Prostaglandin E2 differentially inhibits airway fibroblast migration and contraction in a specific, dose-dependent manner. Conclusions: PGE2 is activated during mucosal inflammation and acts to decrease fibroplastic activity in the mucosal wound bed. During subglottic stenosis (SGS) development, the levels of PGE2 generated in response to injury may be insufficient to blunt the intrinsically fibroplastic phenotype of SGS fibroblasts, resulting in excessive scarring. Laryngoscope, 2009 [source] Molecular analysis of the GNPTAB and GNPTG genes in 13 patients with mucolipidosis type II or type III , identification of eight novel mutationsCLINICAL GENETICS, Issue 1 2009M Encarnação Mucolipidosis II (ML II) and mucolipidosis III (ML III) are diseases in which the activity of the uridine diphosphate (UDP)- N -acetylglucosamine:lysosomal enzyme N -acetylglucosamine-1-phosphotransferase (GlcNAc-phosphotransferase) is absent or reduced, respectively. In the absence of mannose phosphorylation, trafficking of lysosomal hydrolases to the lysosome is impaired. In these diseases, mistargeted lysosomal hydrolases are secreted into the blood, resulting in lysosomal deficiency of many hydrolases and a storage-disease phenotype. GlcNAc-phosphotransferase is a multimeric transmembrane enzyme composed of three subunits (,, , and ,) encoded by two genes ,GNPTAB and GNPTG. Defects in GNPTAB result in ML II and III whereas mutations in GNPTG were only found in ML III patients. We have performed a molecular analysis of the GNPTAB and GNPTG genes in 13 mucolipidosis II and III patients (10 Portuguese, one Finnish, one Spanish of Arab origin and one Indian). Mutations were identified by the study of both cDNA and gDNA. The GNPTAB and GNPTG mRNA expressions were determined by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). The study led to the identification of 11 different mutations. Eight of these mutations are novel, six in the GNPTAB gene [c.121delG (V41FfsX42), c.440delC (A147AfsX5), c.2249_50insA (N750KfsX8), c.242G>T (W81L), c.1208T>C (I403T) and c.1999G>T (p.E667X)] and two in the GNPTG gene [c.610-1G>T and c.639delT (F213LfsX7)]. With regard to the mRNA expression studies, the values obtained by qRT-PCR indicate the possible existence of feedback regulation mechanisms between ,/, and the , subunits. [source] DNER modulates adipogenesis of human adipose tissue-derived mesenchymal stem cells via regulation of cell proliferationCELL PROLIFERATION, Issue 1 2010J.-R. Park Objectives:, In recent years, obesity has become a global epidemic, highlighting the necessity for basic research into mechanisms underlying growth of adipose tissue and differentiation of stem cells into adipocytes, in humans. For better understanding of cell signalling in adipogenesis, the role of DNER (delta/Notch-like EGF-related receptor) in adipogenic differentiation of human adipose tissue-derived mesenchymal stem cells (hAMSC) was investigated. Materials and methods:, To assess the role of DNER in hAMSC adipogenesis, hAMSCs were transfected with DNER small interfering RNA (siDNER). Real-time quantitative reverse transcriptase polymerase chain reactions to assess expression levels of adipogenesis-related genes regulated by siDNER, cell cycle and immunoblot analyses were performed. Results:, First, it was determined that DNER mRNA was profoundly expressed in hAMSCs and reduced during adipogenic differentiation. Knockdown of DNER altered cell morphology, inhibited proliferation and increased frequency and efficiency of adipogenesis in hAMSC. Expression of CCAAT/enhancer-binding protein , increased and proportion of cells in S phase decreased by knockdown of DNER, using specific siRNA. Moreover, adipocyte-specific genes including peroxisome proliferator-activated receptor gamma, fatty acid binding protein 4 and perilipin were up-regulated in siDNER compared to the siControl group during adipogenesis in hAMSC. Conclusions:, These results indicate that DNER knockdown in hAMSC accelerated onset of adipogenic differentiation by bypassing mitotic clonal expansion during the early stages of adipogenesis. [source] | |||||||||||||