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Mast Cell Tryptase Controls Paracellular Permeability of the Intestine 生科四甲 陳子甯
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作者假設: mast cells signal to colonocytes by release of tryptase and activation of PAR2, and that PAR2 couples to ERK1/2 by a βARR- dependent mechanism to regulate TJs. ERK1/2 : extracellular signal-regulated kinases 1/2 arrestin βARR:β-arrestin TJ: tight junction
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作者目標: 證實 PAR2 的活化造成 TJs 和細胞膜滲透性的改變。 證實 mast cell 藉由分泌 tryptase 來活化 PAR2 ,造成 colonocytes 細胞 內鈣離子濃度上升及細胞膜滲透性提高。. 證實 ERK1/2 及 βARR 在 PAR2 活化的這個機制中,扮演何種角色 ERK1/2 : extracellular signal-regulated kinases 1/2 arrestin βARR:β-arrestin
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Mast cell:
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Model for β-arrestin-dependent activation of ERK1/2 by PAR2 microtubule-associated proteins (MAPs) phospholipase A2 (PLA2) Ca 2+
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FIG. 1. Expression of mRNA encoding PAR2 and trypsinogen IV by colonocytes. 525bp 400bp 525bp 400bp c p c T c p cT 確認 NCM460 和 T84 這兩株小腸表皮細胞都能夠表現 PAR2 和 trypsinogenIV NCM460 和 T84 都是種小腸表皮細胞
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FIG. 2. PAR2-mediated mobilization of [Ca 2+ ] i in colonocytes. AP: 由六個 AA 組成 -SLIGKV RP: 是 AP 序列相反的 peptide-VKGILS 。用來當 做 control 組。 結論:如果細胞外無鈣離子,細 胞仍可利用本身所儲存的鈣離子 來進行訊號傳遞。
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FIG. 3. PAR2-mediated mobilization of [Ca 2+ ] i in colonocytes. 結論: trypsin 活化 PAR2 後,的確會讓細胞體內的鈣離子增加。 而 trypsin 的確對 PAR2 有專一性。 c
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FIG. 4. Characterization of CHMC. 確認 mast cell 與 IgE binding 後,可促進 degranuled 。 測不同型態的 CHMC 的 螢光強度。 CHMC 是種 mast cell SBTI: Soybean trypsin inhibitor 證實 mast cell 分泌 tryptase 的方式是 degranule 。
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Flow Cytometry
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FIG. 5. Mast cell signaling to colonocytes. 48/80 :是種促使 mast cell degranule 的藥物。 證實 mast cell 的確是用 degranule 的方式將 tryptase 送至 supernatant form 中,傳至 colonocytes
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TER ( transepithelial resistance ) & paracellular permeability
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FIG. 6. Effects of PAR2 agonists on paracellular permeability. 證實 PAR2 Activation Increases Paracellular Permeability HRP 是種位於細胞質中的酵素。
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ZO-1 與 E-cadhesin Apical Basal Zo-1 E-cadhesin
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FIG. 7. PAR2-induced redistribution of TJ proteins and F-actin detected by microscopy ZO-1 Claudin-1 F-actin occludin Isotype 證實了 PAR2 的確會讓 TJ protein 和 F-actin redistribution 。
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FIG. 8. PAR2-induced redistribution of TJ proteins detected by subcellular fractionation and Western blotting. 結論: PAR2 的確 會造成 TJ proteins 的 redistribution E-cadherin is a transmembrane glycoprotein that mediates epithelial cell-to-cell adhesion.
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FIG. 9. Effects of mast cell products on paracellular permeability. 證實了 PAR2 的確會改變 paracellular permeability
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FIG. 10. ERK1/2-mediated changes in paracellular permeability. ERK1/2 可以調控 paracellular permeability , 而 PAR2 可以調控 ERK1/2 ,所以 PAR2 與調 控 paracellular permeability 有關。 UO126 :是種抑制 ERK1/2 磷酸化的藥劑。
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FIG. 11. Down-regulation of βARR using siRNA βARR1 βARR2 498bp 381bp βARR 是種 cytoplasm-nucleus messenger , 會與 PAR2 結合,活化 ERK1/2 證實: βARR 會活化 ERK1/2
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FIG. 12. βARR-mediated changes in paracellular permeability. Oligofectamine 可促進 βARR 與 ERK1/2 binding 的一種藥劑。 證實 PAR2 活化後,使 βARR 促進 ERK1/2 活化, 進而改變 colonocytes 的滲透性。
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Conclusion Colonocytes expressed PAR2 mRNA and responded to PAR2 agonists with increased [Ca 2+ ] i Mast cells signal to colonocytes in a paracrine manner by release of tryptase and activation of PAR2. βARR 促進 ERK1/2 的活化,導致 colonocytes 的 permeability 上升
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