張震東Chang, Geen-Dong臺灣大學:生化科學研究所蔡秉村Tsai, Ping-TsunPing-TsunTsai2010-05-042018-07-062010-05-042018-07-062008U0001-2907200815184200http://ntur.lib.ntu.edu.tw//handle/246246/178853細胞膜的破裂，對於處在正常生理所產生的外力情況下的細胞，譬如心肌或者肌肉細胞， 是常見的細胞損傷之ㄧ 。目前認為鈣離子活化的胞吐作用（exocytosis）使細胞能夠利用胞器的內膜（endomembrane）暫時修補破損的細胞膜。除了許多現象的描述之外，對於這個領域的詳細分子機制多年來一直沒有重大進展，主要因為缺乏一套製造大規模細胞膜損傷的細胞群之方法學。在本篇研究中，我們改良McNeil 與Warder 在1987年發展的beads loading 技術，利用直徑1毫米的磁珠造成大量細胞的細胞膜受損，進而進行大量分析的實驗。在細胞膜損傷後，我們看到了許多蛋白質在量的增加，其中包含了與內質網壓力（ERstress）相關的chaperones。我們更進一步發現p38 MAPK 的參與，以及其上游分子MEK3/6 與ASK-1 的活化。這些結果顯示，細胞膜破損對於細胞來說是另一種壓力形式，細胞並且透過活化p38 MAPK 的訊息傳遞路徑來促進其存活。利用這新的Magnetic beads rolling assay 方法學，我們能夠進一步解析細胞膜受損以及修補，這個常見且重要的生物現象的運作方式。Plasma membrane disruption is a common type of cell injury for cells enduring physiologically generated mechanical forces, such as epithelia and skeletal muscle cells.epair of torn cell surface is essential to cell survival and has been proposed to be mediated by calcium-elicited gel barrier formation and exocytosis. However, the exact mechanisms underlying membrane repair remain still obscure for years owing to the lack of methods to induce large-scale mechanical rupture of cell membrane in vitro.erein we characterize a “magnetic beads rolling” technique modified from the beadloadingethod (McNeil and Warder 1987) for quantitative analyses. Pilot experimentsuggested the involvement of CaMK in this process and protein levels of somenteresting targets were markedly changed during resealing process including severalR stress-related molecules. Importantly, we found that p38 MAPK and its upstreamEK3/6 and ASK-1 were transiently activated and inhibition of p38 impairedmembrane repair. We also observed compensatory endocytosis after membrane rupture.hese results suggest that membrane disruptions employ similar mechanism as othertress responses through p38 MAPK pathway. Equipped with this newly developedssay, we are in the processes of dissecting the signal transduction pathway involved inembrane rupture and repair.iiibstract (Chinese)……….…………..…………………………………………………ibstract……………………………...………………………………………………...iiontents………..……………………..………………………………………………iii. Introduction………………………...………………………………………………..1. Cell membrane is easily hurt……………….…………………………………...1. Resealing is essential and rapid…….…………………………………………...1. Resealing requires calcium dependent exocytosis……………………………...2. The patch hypothesis……………………………………………………………3. The sealing organelles…………………………………………………………..4. The proteins involved…………………………………………………………...5. Strategies for cell wounding assays……………………………………………..5I. Materials and methods……….……………………………………………………..7. Cell culture……………………………………………………………………...7. Cell wounding assay……….……………………………………………………7. Gel electrophoresis and western blot analysis………………….……………….8. Cloning and purification of the recombinant Annexin a2……….……………...9. Preparation of polyclonal anti-Annexin a2 antibodies………………………...10. Cell viability…………………………………………………………………...10. RT-PCR………………………………………………………………………..11. Immunofluorescence…………………………………………………………..11. Statistical analysis……………………………………………………………..11II. Results……………………………………………………………………………12. Magnetic beads rolling assay and cell line decision…………………………..12. Beads to cell density test………………………………………………………13. Characteristics of beads-rolled cells……………………………………………13. Calcium signaling in membrane resealing……………………………………...14. Up-regulation of stress response proteins………………………………………15. p38 MAPK-mediated signals are required for post-wounding survival………..16. p38 activation affects related proteins stability………………………………...16. Upstream regulation of p38 signaling in A431-III during membrane repair.. …17. Protein localization after wounding………………………………………….…180. Observation of endocytosis in post-wounding cells……………..…………....19V. Figures and tables………………………………………………………………...21. Discussion………………………………………………………………………...30. Membrane disruption causes stress and activates p38 signaling……………….30. Protein stabilization by p38 signaling………….……………………………….30. Signaling module of membrane repair.…………………………………………31. Misfold protein response………………………………………………………..32. Future challenges in magnetic beads rolling……………………………………33I. References………………………………………………………………………..34II. Supplementary figures and tables……………………………………………….40application/pdf10663190 bytesapplication/pdfen-US細胞膜損傷細胞膜修補內質網壓力p38 MAPK磁珠Membrane repairstress responsebeads rollingexocytosishttp://ntur.lib.ntu.edu.tw/bitstream/246246/178853/1/ntu-97-R95b46016-1.pdf