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In the present study, the pattern of cyclooxygenase-2 (COX-2) expression in health and inflamed porcine uteri was analyzed using real-time reverse transcriptase-polymerase chain reaction (RT-PCR),Western blot and immunohistochemistry. On day 3 of the estrous cycle, 50 ml of saline or 50 ml of Escherichia coli (E. coli) suspension containing 10⁹ colony-forming units/ml, were injected into each uterine horn of the control (n=6) and experimental gilts (n=7), respectively. This latter procedure lead to a moderately (n=3) or severely intense (n=4) acute endometritis after eight days. Expression of both the COX-2 mRNA and protein was increased in the endometrium (ENDO) of animals suffering from the moderate (P<0.05, P<0.01, respectively) and severe (P<0.01) acute endometritis, as compared to the control tissues. Moreover, COX-2 mRNA level and protein content were higher (P<0.05) in the ENDO of animals with severe than with a moderately acute endometritis. An elevation in the COX-2 gene (P<0.05) and protein (P<0.001) expression was also observed in the myometrium (MYO) of animals suffering from severe endometritis, when compared with the levels observed in MYO of both the health and moderate intensely inflamed uteri. However, both the COX-2 mRNA and protein levels were similar in MYO of the control and moderately inflamed organs. The luminal epithelium, some of uterine glands and circular layer of the MYO were more intensely stained for COX-2 in animals with severe endometritis, than in animals with healthy or moderately inflamed uteri. Nonetheless, stronger COX-2 reaction was found in some of the uterine glands in latter group, when compared to that observed in uteri of the control animals. While positive COX-2-labeling was observed in the muscular layer of all arteries supplying the health and inflamed uteri, such staining was exclusively present in the endothelium of some arteries in inflamed organs. Likewise, some arteries in uteri of the animals with severe endometritis displayed immunoreaction stronger than that found in uteri of the animals with moderate inflammation. The present study revealed an up-regulation of COX-2 mRNA and protein in the inflamed porcine uterus, which was directly related to the intensity of the organ inflammation. An increase in the COX-2 expression in the uterus challenged by E. coli-induced inflammation indicates that this enzyme is crucial for elevated prostaglandins production in the inflamed organ.
Application of real-time RT-PCR (rRT-PCR) for detection of swine vesicular disease virus (SVDV) in samples of archival SVDV isolates and clinical samples collected from SVDV infected pigs was described. A primer set that targets the IRES region of the SVDV genome and TaqMan probe specific for a highly conserved region in SVDV RNA IRES region were used. The assay detected viral RNA in all tested archival strains of SVDV isolated in Europe during years 1972-73 and 1992 as well as in clinical samples collected from experimentally infected pigs. The rRT-PCR can provide quantitative and qualitative information and is more sensitive and faster to perform than the conventional RT-PCR.
A real-time reverse transcription-PCR (rRT-PCR) for the detection of the foot-and-mouth disease virus (FM DV) in samples of archival isolates of FMDV serotypes O, A, C, SAT1-3, and Asia 1 was described. A primer set that targets the IRES region of the FMDV genome and TaqMan probe specific for a highly-conserved region in FMDV RNA IRES region was used. The assay detected the viral RNA in all tested archival serotypes of FMDV. Swine vesicular disease virus (SVDV) and bluetongue virus (BTV) RNA as well as RNA isolated from epithelium taken from healthy uninfected calf (negative control) were undetectable in the test. The detection limit of rRT-PCR was quantified for 1 TCID₅₀. The test can provide quantitative as well as qualitative information and is more sensitive and much faster to perform than the conventional RT-PCR. It can be used in large-scale screening because of its ability to simultaneous analysis up to 96 samples per run. The applied rRT-PCR is accurate, specific, and reliable technique for the detection of FMDV in biological samples. Therefore it is seen as a valuable tool to complement the routine diagnostic procedure for FMD virus diagnosis.
Statins are inhibitors of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), the key enzyme of the sterol biosynthesis pathway. Statin therapy is commonly regarded as well tolerated. However, serious adverse effects have also been reported, especially during high-dose statin therapy. The aim of our study was to investigate the effect of statins on gene expression profiles in human hepatoma HepG2 cells using Affymetrix Human Genome U133 Plus 2.0 arrays. Expression of 102, 857 and 1091 genes was changed substantially in HepG2 cells treated with simvastatin, fluvastatin and atorvastatin, respectively. Pathway and gene ontology analysis showed that many of the genes with changed expression levels were involved in a broad range of metabolic processes. The presented data clearly indicate substantial differences between the tested statins.
Several studies have reported tumour infiltrating CD4+ T cells as a favourable prognostic factor in some types of cancer. We investigated 37 head and neck squamous cell carcinomas (HNSCC) at different stages, using immunohistochemical staining for CD4+ infiltrates and real-time reverse transcription polymerase chain reaction (RT-PCR) detection of CD4 mRNA. The CD4+ infiltrates were evaluated and expressed as a percentage according to the ratio of CD4+ T cells to epithelial cells in the cancer cell nests and to the overall inflammatory cell infiltrate in the tumor stroma. The CD4 mRNA expression level strongly correlated with the CD4+ infiltration score in the cancer epithelium (rs = 0.858, P < 0.001) and in the cancer stroma (rs = 0.797, P < 0.001). These results indicate that the real-time RT-PCR assay is a sensitive and reliable method for the detection of CD4 mRNA, and that it could be used to reassess CD4+ infiltration status in resected specimens from patients with HNSCC.
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