사람 Telomerase 역전사효소 서열 유래 펩타이드 GV1001의 항염 효능에 대한 연구

Abstract

학위논문 (석사)-- 서울대학교 대학원 : 의학과(해부학 전공), 2015. 8. 강재승.GV1001 is a peptide derived from human telomerase reverse transcriptase (hTERT) sequence, and has anti-cancer and anti-inflammatory effect. Enolase1 (ENO1) is a glycolytic enzyme and its stimulation induces to produce a large amount of pro-inflammatory cytokines from concanavalin (Con) A -activated peripheral blood mononuclear cells (PBMCs) and from ENO1 expressing monocytes and macrophages from rheumatoid arthritis (RA) patients. However, it is still unknown whether GV1001 could regulate the ENO1-mediated pro-inflammatory cytokines production. Therefore, I investigated whether GV1001 regulates ENO1-mediated pro-inflammatory cytokines production as an anti-inflammatory peptide. First, I found that GV1001 does not affect the expression of ENO1 on Con A-activated PBMCs and RA PBMCs. However, ENO1 stimulation increased the production of pro-inflammatory cytokines (TNF-α, IL-1β and IL-6) from Con A-activated PBMCs. And it is down-regulated by the pre-treatment of GV1001. GV1001 also decreases the production of pro-inflammatory cytokines from ENO1 stimulated RA PBMCs. And then I examined what kinds of signaling molecules are involved in the down-regulation of ENO1-mediated pro-inflammatory cytokine production by GV1001. When ENO1 on the surface of Con A-activated PBMCs and RA PBMCs is stimulated, I found that p38 MAPK and NF-κB are activated. However, these are successfully suppressed by the pre-treatment of GV1001. Taken together, GV1001 might be a useful anti-inflammatory peptide via the down-regulation of pro-inflammatory cytokine production and the suppression of the p38 MAPK and NF-κB activation by ENO1 stimulation.CONTENT Abstract ............................................................... ⅰ Contents .............................................................. ⅳ List of Figures ................................................... ⅶ List of Abbreviations ........................................ ⅹ Introduction ......................................................... 1 Materials and Methods 1. Isolation of PBMCs .................................................................... 5 2. Stimulation of PBMCs with Con A .......................................... 5 3. Preparation of Anti-ENO1 monoclonal antibody.................... 6 4. ENO1 stimulation ....................................................................... 7 5. GV1001 treatment ..................................................................... 7 6. Flow cytometry analysis .......................................................... 7 7. Enzyme-Linked Immunosorbent Assay (ELISA) ................. 8 8. Inhibitor study for signal pathway ........................................ 9 9. Immunoblotting ........................................................................... 9 10. Statistical analysis ................................................................. 10 Results 1. Con A stimulation increases ENO1 expression on the surface of PBMCs ................................................................................................... 12 2. GV1001 doesn't affect ENO1 expression on PBMCs by Con A treatment ................................................................................................... 16 3. GV1001 decreases the production of TNF-α, IL-1β and IL-6 by ENO1 stimulation ................................................................................................... 18 4. GV1001 suppresses the activation of p38 MAPK and NF-κB in Con-A activated PBMCs: ELISA ................................................................................................... 22 5. GV1001 suppresses the activation of p38 MAPK and NF-κB in Con-A activated PBMCs: Immunoblotting ................................................................................................... 25 6. GV1001 doesn't change the expression level of ENO1 on PBMCs from RA patients ................................................................................................... 31 7. GV1001 decreases the production of TNF-α, IL-1β and IL-6 from RA PBMCs by ENO1 stimulation ................................................................................................... 34 8. GV1001 suppresses the activation of p38 MAPK and NF-κB in RA PBMCs. ................................................................................................... 37 Discussion .......................................................... 39 References ......................................................... 44 Abstracts in Korean ......................................... 52Maste