VIASAN Programme - Project 196
VIASAN Programme (National Plan for Research-Development and Innovation) Project 196 (2003-2005): Cellular Model for Study of the Pathogenesis of Pulmonary Fibrosis
Coordinator: University of Bucharest
Project director: prof. Dana Iordăchescu, PhD
Team members: prof. Otilia Zărnescu, PhD, lect. Anișoara Cîmpean, PhD; PhD students: Adela Bonoiu, Gabriela Bucată, Raluca Ciubăr, Laura Homeghiu, Cristina Iancu, Cristina Stan; Master student: Valentina Mitran
Partners: University Emergency Hospital, Bucharest, Clinical Laboratory of Pathological Anatomy (prof. Maria Sajin, PhD)
The project integrates into the general objectives of the VIASAN programme of promotion and supporting of multidisciplinary researches, in order to understand the pathogenesis of diseases with high impact within population and their treatments, in the European context, for the dissemination of the Romanian biomedical research.
The project is enclosed in thematics of the Subprogramme 3 «Molecular and cellular medicine», concerning the study of some cellular processes in biology and pathology, since the pulmonary fibrosis is the final common pathway of a diverse group of lung disorders, of unknown etiology, that lead to progressive and irreversible changes in the lung architecture and for that the treatment to date has proved largely ineffective. The project relies on the hypothesis that the primary event of the pulmonary fibrosis is the injury/activation of the alveolar epithelial cells that synthesize a variety of cytokines and growth factors, involved in migration, proliferation and fibroblast phenotype alteration, in extracellular matrix remodeling and then in the destruction of the lung parenchyma – key elements in the transformation of a reversible process in a progressive and irreversible one. In the pathogenesis of the pulmonary fibrosis can be also involved the reactive oxygen species (ROS), especially in the case of the professional maladies, that appear as a consequence of the inhalation of some metal particles, being known the relationship between the signaling pathways of ROS and profibrinogenic cytokines.
The cellular model of study of the pulmonary fibrosis has been achieved by a comparative study of the results regarding the treatment of the human lung fibroblasts with TGF-b1 or/and cadmium chloride. TGF-b1 is a key pro-fibrotic mediator, a chemotactic agent for fibroblasts that promotes their transformation into miofibroblasts (connective cells that grow in muscles and can produce fibrosis), that induce the synthesis of the extracellular matrix proteins and inhibit collagen degradation by TIMP induction and a decrease of the matrix metalloproteinases’ level. Cadmium (Cd2+) is a heavy metal widely spread in the environment, particularly toxic for human, being involved in the pathogenesis of some professional maladies, in the development of emphysema and of the pulmonary fibrosis. Cd2+ toxicity implies oxidative mechanisms because the antioxidant therapy diminishes it.
The precise objectives of the project:
The comparative study of human pulmonary fibroblasts isolated from regions with fibrotic foci and from normal pulmonary tissue;
Development of a cellular model that reproduces the morphological and biochemical changes of fibroblasts during pulmonary fibrosis;
Verification of the cellular model by biochemical, cellular and molecular comparative studies on pulmonary fibroblasts – normal, isolated from fibrotic foci and experimental induced pulmonary fibrosis;
Study of the action of some anti-fibrotic compounds upon the cellular model.
Activities for fulfilment of the project objectives:
Histopathological analysis and selection of the anatomical pieces for fibroblast isolation;
Obtaining of the cell culture;
Studies of cellular phenotype, viability, proliferation, cytotoxicity;
Cell treatment with TGF-b /+ CdCl2;
Study of the oxidative stress;
Estimation of the pro-inflammatory cytokines secreted in the medium;
Study of the matrix metalloproteinase expression and activity;
Study of the KGF (keratinocyte growth factor) activity.
The material base:
Animal cell culture laboratory with sterile areas (laminar-flow hood, CO2 incubator, inverted microscope + photo-documentation system + epi-fluorescence system, refrigerated centrifuge and micro-centrifuge, balance, vortex), Laboratory for material processing (systems of humidified and dry sterilization, balances), Station for water purification (equipment for obtaining the ultrapure water Milli Q Synthesis), Laboratory for cell cryopreservation (deep freezer at –96oC, liquid nitrogen cryostorage containers) and freezing room, Laboratory of Biochemistry (UV/VIS spectrophotometers, spectrofluorimeter, refrigerated centrifuge, electrophoresis and western-blot systems, UV/VIS transiluminator, thermostated water baths, analytical balances, magnetic stirrers) and Multi-user Research Center of Molecular Biology (micro-plate reader TECAN, image computerized densitometer, spectrophotometer).
