Tabacco as a Cultural Phenomenon in the Czech Republic

katedra: KFL

The effect of growth factors on gene expression in rat liver myofibroblasts

Jaterní tkáň je tvořena několika typy buněk. Při poranění jater dochází k fibrotizaci nekrotické tkáně, pomocí aktivovaných jaterních hvězdicovitých buněk (HSC) a portálních myofibroblastů. V této práci jsme sledovaly vliv kultivačního prostředí v kombinaci s růstovým faktorem FGF-1 na expresi určitých genů v jaterních myofibroblastech. V první fázi experimentu jsme se zaměřily na srovnání exprese genů v buňkách kultivovaných v dvojrozměrném (2D) prostředí na polystyrenových Petriho miskách s kultivací v trojrozměrném (3D) kolagenním gelu. V obou případech po dvanácti hodinové expozici růstovým faktorem FGF-1. V druhé fázi pokusu jsme se zaměřili na interakci FGF-1 s heparinem. Buňky jsme získali z frakce neparenchymových jaterních buněk potkanů pomocí opakovaného pasážování. Buňky jsme kultivovaly v kolagenním gelu a na plastu v kultivačním médiu s přídavkem růstového faktoru. Po kultivaci jsme z buněk izolovali celkovou RNA, která byla převedena pomocí reverzní transkriptázy na cDNA. Expresi genů jsme stanovili pomocí rtRT-PCR. Data jsme statisticky zpracovali pomocí Studentova t-testu. Z experimentu vyplynulo, že kultivační prostředí má zásadní vliv na kultivované myofibroblasty a že FGF-1 ovlivňuje expresi genů v jaterních myofibroblastech. Naše práce přinesla i poznatek, že exprese genů buněk kultivovaných s FGF-1 a buněk kultivovaných s FGF-1 a heparinem, se liší. Zaměřili jsme se na změny v expresi genů matrix metaloproteinázy 9, matrix metaloproteinázy 13 a osteopontinuThe liver tissue consists of several types of cells. Liver injury leads to fibrosis of the necrotic tissue induced by hepatic stellate cells (HSC) and portal myofibroblasts. We have studied the influence of the fibroblast growth factor FGF-1 on the expression of certain genes in hepatic myofibroblasts. In the first part of the experiment, we compared the gene expression in cells growing on polystyrene Petri dishes (2D culture) and in cells embedded in collagen gel(3D culture), after a 12-hour exposition to the growth factor FGF-1. In the second part, we studied the interaction between FGF-1 and heparin. The cells were isolated from the fraction of non-parenchymal liver cells of Sprague- Dawley rats by repeated passaging. After harvesting the cells total cellular RNA was isolated and translated into cDNA using the reverse transcriptase. We used rtRT-PCR to evaluate gene expression. We focused on the changes of gene expression of matrix metalloproteinase 9, matrix metalloproteinase 13 and osteopontin. The data was statistically analysed by Student´s t-test. The experiments showed that the cellular environment has crucial influence on hepatic myofibroblasts in vitro and that FGF-1 affects gene expression in these cells. The largest changes were found in the expression of matrix metaloproteinase 13.Katedra biologických a biochemických věd1. Prezentace výsledků diplomové práce. 2. Diskuze k posudkům vedoucího a oponenta diplomové práce. 3. Diplomantka zodpověděla všechny dotazy a připomínky k DP.Dokončená práce s úspěšnou obhajobo

The Influence of Extracellular Matrix Components to Cells Cultured In Vitro

Myofibroblast expansion is a critical event in the pathogenesis of liver fibrosis. The activation of hepatic stellate cells (HSC) to myofibroblast (MFB) results in the enhanced production of extracellular matrix (ECM). We have studied the effect of fibroblast growth factor 1 (FGF-1) on liver MFB. In the second part we investigated effect of transforming growth factor β1 (TGF-β1) and FGF-1 on cell line HSC-T6. Cells were cultured on plastic dishes and in 3D collagen gel mimicking fibrotic tissue. MFB were isolated by repeated passaging of nonparenchymal liver cell fraction. The transfer of MFB from plastic dishes to collagen gel resulted in the change in their shape and phenotype. The expression of cytokine TGF-β1 and of MFB markers, α-smooth muscle actin (α-SMA) and cellular fibronectin (EDA-FN) on protein level was significantly decreased in collagen gel. The experiments with SB 431542, the inhibitor of TGF-β receptor type I, showed that EDA-FN and α-SMA are differently regulated. EDA-FN expression is dependent on TGF-β1, while the expression of α-SMA is primarily determined by the environment and modified by TGF-β1. EDA-FN is more sensitive to the U0126, the inhibitor of protein kinases MEK 1 and 2. Collagen gel does not change the expression of metalloproteinase MMP-2 but activates the proenzyme...

Fibroblast Growth Factor-1 Suppresses TGF-β-Mediated Myofibroblastic Differentiation of Rat Hepatic Stellate Cells

Myofibroblast expansion is a critical event in the pathogenesis of liver fibrosis. The activation of hepatic stellate cells (HSC) to myofibroblast (MFB) results in the enhanced production of extracellular matrix (ECM). In this study, we explored the effect of acidic fibroblast growth factor (FGF-1) treatment on a transforming growth factor (TGF-β1) induced MFB conversion. We used HSC-T6 cell line, which represents well-established model of activated HSC. These cells strongly expressed α-smooth muscle actin (α-SMA) and fibronectin (FN-EDA) after stimulation with TGF-β1, which is a stimulus for MFB differentiation and ECM production. FGF-1 reduced proteins expression to levels comparable with untreated cells. Mild repression of secreted gelatinases was seen in culture media after FGF-1 treatment. The exposure of cells to collagen gel leads to changes in cell morphology and in expression of MFB markers. Lack of α-SMA in cells embedded to collagen gel was detected. When stimulated with TGF-β1, the cells increased expression of FN-EDA, but not α-SMA. Although the cells on plastic and in collagen gel show different properties, FGF-1 reduced expression of FN-EDA in both conditions. Disrupting TGF-β1 signalling pathway represents a potential strategy for the treatment of fibrosis. We showed that FGF-1 could antagonize signals initiated by TGF-β1

Exhaled Breath Condensate: Pilot Study of the Method and Initial Experience in Healthy Subjects

Analysis of Exhaled breath condensate (EBC) is a re-discovered approach to monitoring the course of the disease and reduce invasive methods of patient investigation. However, the major disadvantage and shortcoming of the EBC is lack of reliable and reproducible standardization of the method. Despite many articles published on EBC, until now there is no clear consensus on whether the analysis of EBC can provide a clue to diagnosis of the diseases. The purpose of this paper is to investigate our own method, to search for possible standardization and to obtain our own initial experience. Thirty healthy volunteers provided the EBC, in which we monitored the density, pH, protein, chloride and urea concentration. Our results show that EBC pH is influenced by smoking, and urea concentrations are affected by the gender of subjects. Age of subjects does not play a role. The smallest coefficient of variation between individual volunteers is for density determination. Current limitations of EBC measurements are the low concentration of many biomarkers. Standardization needs to be specific for each individual biomarker, with focusing on optimal condensate collection. EBC analysis has a potential become diagnostic test, not only for lung diseases