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Regulation of cell death mode from apoptosis to necroptosis
Korea (KMSA) - Chungnam university, Daejeon
Department of Pharmacology, College of Medicine
Gang Min Hur
Gang Min Hur, M.D., Ph.D.
Type of Research Project
- Basic science
What is the background of the project?
Tumor necrosis factor (TNF) is a pleiotropic cytokine that mediates diverse physiological events including nuclear factor kappaB (NF-κB) activation, cell survival and cell death, in the form of apoptosis and necroptosis. Importantly, both types of cell death occurs in vivo not only during normal physiological processes but also in pathological conditions such as myocardial ischemia, stroke and TNF-induced inflammatory response syndrome. Thus, discovery of compounds that regulate the apoptotic and necroptotic pathway will be of important value in developing therapeutic approach for relevant diseases.
What is the aim of the project?
To identify the noevl compounds or genes regualting apoptosis or necroptotic cell death upon DR ligation.
What techniques and methods are used?
1. Cell culture and transfection: A suitable cancer cells will be cultured in Dulbecco’s modified Eagle’s medium (DMEM) including 10% fetal bovine serum, 2 mmol/L glutamine and 100 U/mL penicillin/ streptomycin in 5% CO2 atmosphere at 37°C. Transient transfections of some expression plasmids will be performed using Lipofectamine 2000. 2. Cell viabilty assay: Cells will be plated onto 96 well plates at different cell densities, depending on cell type. After treatment the cells with some ligands, cell viability assay will be conducted utilizing Cell Titer-glo Luminescent Cell Viability Assay kit which measures cell viability based on adenosine triphosphate (ATP) levels present in live cells. Luminescent measurements were taken on a Tecan Infinite Plate Representative images were also taken by an inverted microscope. 3. Immuno blot assay and cell death mode identification by apoptotic and necroptotic markers: After treatment with different conditions, cells will be collected and lysed in M2 buffer (20 mM Tris, pH 7.6, 0.5% NP-40, 250 mM NaCl, 3 mM EDTA, 3 mM EGTA, 2 mM dithiothreitol, 0.5 mM PMSF, 20 mM β-glycerol phosphate, 1 mM sodium vanadate, and 1 µg/ml leupeptin). Fifty micrograms of cell lysates will be fractionated by 10% or 6% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and visualized by enhanced chemiluminescence. 4. Statistical analysis: The difference between groups will be analyzed by Student's t-test using SPSS sofetware ver. 13.0 (SPSS, Inc)
What is the role of the student?
- The student will mainly observe
- The tasks of the student will be performed on his/her own
- The tasks will be done under supervision
What are the tasks expected to be accomplished by the student?
During the time in the lab, the students shoud learn some experimental techniques as follows: 1. The principle and practice of cell culture. 2. The principle and practice of molecular cloning of the interesting genes: insertion of interesting genes into expression plamids,.isolation and purification from E Coli, Digestion of plasmids with DNA restirction enzyme et al., 3. The knowledge of signaling pathway for apoptosis and necroptosis. 4. The knowledge of escape mechanism of cancer cells against cell death
Will there be any theoretical teaching provided (preliminary readings, lectures, courses, seminars etc)
What is expected from the student at the end of the research exchange? What will be the general outcome of the student?
- No specific outcome is expected
What skills are required of the student? Is there any special knowledge or a certain level of studies needed?
Are there any legal limitations in the student’s involvement
Type of students accepted
This project accepts: - Medical students
- Balkwill; F. (2009) Tumour necrosis factor and cancer. Nat Rev Cancer 9:361-371.
- Brenner; D.; Blaser; H.; and Mak; T. W. (2015) Regulation of tumour necrosis factor signalling: live or let die. Nat Rev Immunol 15; 362-374.
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