Ruolo biologico dell' ormone lattogeno placentare nel differenziamento della cellula beta pancreatica
Author(s)
Bartolini, Barbara
Date Issued
October 24, 2008
Type
Doctoral Thesis
Abstract
Diabetes Mellitus is one of the most widespread pathologies in Western Society and industrialized
countries and it shows a modified insulin secretion from the pancreatic beta cells, with consequent hyperglycaemia. To this very day the treatment for the cure of diabetes is insulin and/or surrogates and ultimately the organ transplants. With regard to this, different protocols have been developed for pancreas transplant as for istance the combined pancreas-kidney transplant that seems to allow a better life expectation. At present the technique of Langherans Islet transplant instead of organ
tranplant is in progress. Moreover organ transplant and Langherans Islet transplant is limited owing to the scarce availability of donors. To overcome this restriction of organ supplies in many research laboratories studies are in progress as to obtain pancreatic beta cells from differentiation of embryonic stem cells, from bone marrow stem cells, or from pancreatic duct stem cells and also from the de-differentiation and trans-differentiation of different cellular types. It has been proved
that pancreatic beta cells can proliferate after numerous physiological and pathophysiological
stimuli. A great number of growth factors have shown to encrease proliferation of beta cells, their mass and their function. Among the growth factors the most important are: the Placental Lactogen Hormone (PL), the Growth Hormone (GH) and the Prolactin (PRL), that belong to a family of polipeptidic homologous hormons that are common to different species from primates to teleostei fish. PL is produced by the placenta exclusively during pregnancy and it represents the most important factor for the increase of the Langherans Islet mass and their function. PL exerts a bigger mitogenous actvity on pancreatic beta cells than GH and PRL, therefore it could play a potential role on the survival and funtion of the Islet during and/or after transplant. Our interest is focused on the possible biological role of the hPL-A hormone in the cell differentiation.
The aim of this PhD thesis is that of optimising a method of expression and purification of the
Human Placental Lactogen Hormone (isoform A) (hPL-A) not commercially available, physiologically active, that can be used in the study of a possible role exerted by hPL-A in the
differentiation of pancreatic duct cells in to beta cells and analyzing the molecular mechanism of signal transduction. The cellular lines PANC-1 (human adenocarcinoma of the pancreas duct) have
been used in this study. The serum depleted cells have been stimulated for 96 hours with 500 ng/ml
of protein purified in our laboratory, following an experimental protocol developped in our
laboratory. The results reported in this thesis show that the hormone hPL-A stimulates the
phosphorylation of the proteins AKT and MAPK (p44-p42), from which is evicted a possible biological role in favouring the signal that induces the survival of pancretaic beta cells; the
immunofluorescence analysis shows that the treated cells change their morphology and express
molecular markers of the pancreatic beta cells (Insulin, Peptide-c; Glut-2, Pdx-1). Moreover
through the proteomics analysis (2D-Gel and DIGE) we have evaluated the change in the proteic
expression associated to the molecular and morphological differentiation shown by the
immunofluorescence analysis. The results obtained suggest that the condition of serum depletion
and/or hormonal stimulation have brought about a change also in the asset of the proteome of the
PANC-1 cells, thus supplying and outline that is coherent with a process of differentiation from
duct tumoral cells to cells with different morphological and functional characteristics.
Additional information
Dottorato di ricerca in Evoluzione biologica e biochimica
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