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PCSK9 expression induced by pro-inflammatory stimuli in mononuclear cells
Universita degli Studi di Pisa
DIP. DI PATOLOGIA CHIRURGICA, MEDICA, MOLECOLARE E DELL'AREA CRITICA Università di Pisa Via Savi 10 56126 PISA (PI), Italy
Prof. Alessandro Celi – Dr.ssa Valentina Scalise
Prof. Alessandro Celi – Dr.ssa Valentina Scalise
Type of Research Project
- Basic science
What is the background of the project?
Proprotein convertase subtilisin/kexin 9 (PCSK9) is a serine protease belonging to the family of PCSKs involved in the activation of peptide hormones and receptors from precursors. By downregulating their intracellular recycling, PCSK9 decreases the number of LDL receptor(R)s available for LDL-cholesterol(C) uptake therefore increasing its circulating levels. The involvement of PCSK9s in atherogenesis, however, spans beyond a mere regulation of circulating LDL-C levels by actively contributing to inflammatory processes tightly connected to innate immunity through several mechanisms including NFκB pathway activation. On the basis of the above summarized background, making it conceivable to hypothesize a mechanistic link between inflammation and PCSK9 expression in immune cells.
What is the aim of the project?
Specific aims of this research proposal will be: 1) To test whether LPS, a well known proinflammatory stimulus, and other mediators such as TNF-alpha, oxLDL, microparticles (MP),are able to stimulate PCSK9 expression in peripheral blood mononuclear cell (PBMC)s and THP-1 cell line; 2) To test the involvement of NFkB in that response by using BAY-117082, a NFκB inhibitor and a specific enzyme-linked immunosorbent assay (ELISA).
What techniques and methods are used?
Cell isolation and culture. Human PBMC suspensions will be obtained from unpooled buffy coats left over from blood bank draws taken from healthy donors and isolated through a discontinuous Ficoll/Hystopaque density gradient. THP-1 cells line will be used to test the direct involvement of monocytes in the PCSK9 expression. PCSK9 protein levels. The expression of PCSK9 will be measured by quantitative sandwich enzyme immunoassay technique (ELISA). NFκB activation. NFκB (p65) will be detected by: 1: A sensitive, non-radioactive method, a 96-well ELISA replaces the cumbersome radioactive electrophoretic mobility shift assay (EMSA); 2: Using a specific NFκB inhibitor, BAY-117082.
What is the role of the student?
- If the project includes “lab work”
- the student will take active part in the practical aspect of the project
- The tasks will be done under supervision
What are the tasks expected to be accomplished by the student?
The tasks that should be performed by the student are the following: 1: Cell isolation and culture 2: Develop hypotheses and propose appropriate experiments to test them. 2: Design and conduct experiments 3: Review experiments in order to evaluate the progress of the research, and ensure experiments conform to established protocols. 4: Use appropriate data analysis and interpret the results of experiments.
Will there be any theoretical teaching provided (preliminary readings, lectures, courses, seminars etc)
The tutor will provide the student articles, reviews and protocols need to understand the background and the specific methods used during the research project.
What is expected from the student at the end of the research exchange? What will be the general outcome of the student?
- The student will prepare a scientific report - No specific outcome is expected
What skills are required of the student? Is there any special knowledge or a certain level of studies needed?
Desire to learn, curiosity, critical thinking and reasoning, determination, teamwork, information handling & organization.
Are there any legal limitations in the student’s involvement
Type of students accepted
This project accepts: - Medical students - Graduated students (less than 6 months) - Pre-Medical students from the American-British system
- 1. Schmidt RJ; Beyer TP; Bensch WR; et al. Secreted proprotein convertase subtilisin/kexin type 9 reduces both hepatic and extrahepatic low-density lipoprotein receptors in vivo. Biochem Biophys Res Commun 2008;370: 634–40.
- 2. Walley KR; Thain KR; Russell JA et al. PCSK9 is a critical regulator of the innate immune response and septic shock outcome. Sci Transl Med. 2014 Oct 15;6(258):258ra143.
- 3. Feingold KR; Moser AH; Shigenaga JK et al. Inflammation stimulates the expression of PCSK9. Biochem Biophys Res Commun. 2008 Sep 19;374(2):341-4.
- 4. Ferri N; Tibolla G; Pirillo A; Cipollone F; Mezzetti A; Pacia S; Corsini A; Catapano AL. Proprotein convertase subtilisin kexin type 9 (PCSK9) secreted by cultured smooth muscle cells reduces macrophages LDLR levels. Atherosclerosis. 2012 Feb;220(2):381-6
- 5. Tang Z1; Jiang L; Peng J; Ren Z; Wei D; Wu C; Pan L; Jiang Z; Liu L. PCSK9 siRNA suppresses the inflammatory response induced by oxLDL through inhibition of NF-κB activation in THP-1-derived macrophages. Int J Mol Med. 2012 Oct;30(4):931-8.
- 6. Li S; Guo YL; Xu RX; et al. Association of plasma PCSK9 levels with white blood cell count and its subsets in patients with stable coronary artery disease. Atherosclerosis 2014; 234: 441–445.
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