Studio di invecchiamento di eritrociti umani, da donatori sani e malati mediante l'utilizzo di DSC, AFM e misure di pattern biochimici

Responsabili di progetto

Marco Girasole, Stefka Taneva

Accordo

BULGARIA - BAS - Bulgarian Academy of Sciences

Bando

CNR-BAS 2016-2018

Dipartimento

Scienze fisiche e tecnologie della materia

Area tematica

Scienze fisiche e tecnologie della materia

Stato del progetto

Nuovo

Proposta di ricerca

Human erythrocytes (red blood cells, RBC) are among the most studied and relatively simple Biosystems, their simplicity arise mainly from the lack of the nucleus, i. e. from their inability of self-replication. In fact the RBC can be seen as a biochemical machine that is assembled, get old and, when the accumulation of damage impairs their functionality, get removed from the circulation (an erythrocyte has an average life cycle of 100 days in the bloodstream). More in detail, the RBC can be considered as optimal indicators of the cardiovascular condition of a person, this consideration arise from their inability to synthetize the single components (as proteins) in response to the environmental conditions that they experience in the bloodstream. These cells, from a scientific point of view, have two major advantages with respect to other cell lines: they are easy to obtain (by taking a blood sample) and easy to purify (by means of centrifugation). Moreover the red blood cells are very easy to be maintained in in-vitro condition, they needs an isotonic buffer enriched with glucose. The fundamental structure of an erythrocyte is the so-called membrane-skeleton, an almost unique architecture. It consists in a dense network of tetrameric polymers of spectrin connected with the lipid double-layer trough the "junctional complexes" and the ankirins. The unbounded parts of the membrane-skeleton appears flexible and allow the dynamic shape modification that the cells experiences in the bloodstream, especially in the narrow capillaries where they can undergo really huge deformation (up to the squeezing of the 50% of the diameter) without the occurrence of fragmentation events. In the junctional complexes resides also the Band-3 protein that plays an essential role in the metabolism of these cells and in the exchange of oxygen between the hemoglobin and the tissues. Alteration to a single component of the membrane-skeleton results in a modification of the whole structure that can compromise the correct functionality of these cells of oxygen carrier in the bloodstream. Atomic Force Microscopy (AFM) studies carried out in our laboratories (citation), in the Institute of Structure of Matter of the CNR, had highlighted the morphological, morphometrical (membrane roughness measurements) and nanomechanical (elasticity measurements) modification that these cells experienced during the ageing process, moreover, we had highlighted how different pathology can affect the structure of the RBCs. Differential Scanning Calorimetry (DSC) studies carried out at the laboratories of professor Germanova (citation), in the INSTITUTE?? of the BAS, on the plasma had highlighted alteration in the protein composition in the plasma of patients affected by different diseases (colorectal cancer, multiple myeloma, gastric cancer).
In this Project our two research groups will study the ageing process of RBC from healthy and pathological situation, to this aim they will combine their specific techniques, AFM and DSC, with the evaluation of several biochemical parameters. The idea is to identify and characterize, by DSC measurements, the proteins mainly involved in the ageing process on three different levels of complexity: whole cells, intracellular content and isolated membrane-skeleton. These information will be coupled with AFM measurements of morpho-metrical and morpho-mechanical parameters of the whole cells and of the isolated membranes by AFM. The obtained data will be correlated with the measure of several biochemical parameters such as cell lysis rate, ATP content, hemoglobin oxidation state, GSH/GSSG ratio etc.... The goal is to find an ageing pattern characteristic of an healthy condition. This very same experimental approach will be applied to the study of RBC obtained from patients with diseases that directly affect the bloodstream, such as diabetes mellitus of type I and II or hypertension). Then the attention will be focused on the study of RBC from cancerous patients, with the aim of finding protein markers that correlates with the pathological condition. The pathological samples will be disposable by an already existing collaboration with the (oncology?? Hematology?? Clinical?? ) of the (Hospital??) in Sofia (Bulgaria).
This project will need the mobility of the involved researchers in each laboratory, in order to homogenize the protocols for the treatment and storage of the erythrocytes and to optimize the sample's preparations protocols. Moreover will be necessary common meeting to discuss the obtained results and to plan the next steps.

Obiettivi della ricerca

This Project will have different objectives:
1- Combine DCS, AFM and biochemical parameters to the study of the RBC's ageing in healthy and pathological donor. The focus will be on the better understanding of the ageing mechanism with particular attention to the mainly involved proteins and the essential morphological marker
2- Validate the DSC as a screening technique for RBCs of donors affected by common cardiovascular disease such as diabetes, arteriosclerosis and hypertension.
3- Find, for the same pathological donors, the peculiar structural anomalies of the RBC by AFM morphometric and morphomechanical characterization of these cells
4- Apply the experimental combined approach to characterize RBC from cancerous patients and find peculiar marker to correlate with the cancer to an early diagnoses of the disease
5- Elaborate a protocol for the treatment and analysis of the samples as well a protocol for the data analysis for an exportation to the daily hospital practice

Ultimo aggiornamento: 08/06/2025