水通道蛋白(aquaporins, AQPs)是細胞膜上的通道蛋白，主要功能是傳遞水或不帶電小顆粒分子，改變細胞內的滲透壓，增加水在細胞膜通透性。在傷口癒合的過程中，AQPs會促進細胞的遷移和增生，提升傷口癒合的效果，並且藉由AQPs極化現象的形成，導致細胞內滲透壓改變，進而調控肌動蛋白(actin)的分布位置和影響重組過程中細胞特性，如細胞形態(morphology)、貼附(adhesion)和接合作用(tight junction)。本研究目的為觀察 AQP3 和 actin 對於細胞遷移和貼附之情形，並且探討傷口癒合過程中，AQP3 極化對於 actin 分布位置和重組過程之影響，建立細胞遷移的模型。
傳統傷口癒合試驗(wound heal assay)結果發現，AQP3 受到氯化鎳抑制會影響細胞遷移，進而降低傷口癒合的能力。傷口癒合過程中，抑制 AQP3 和 actin聚合造成細胞層狀偽足(lamellipodium)消失，表示 AQP3 和 actin 聚合會調控細胞層 狀 偽 足 之 形成 ， 此 與 細 胞 遷 移 與 傷 口 癒 合 有 關 。 以 免 疫 螢光 染 色(immunofluorescence)偵測 AQP3 和 actin 之分布，結果顯示在細胞遷移過程中AQP3 會均勻分布在細胞核周圍，與 actin monomers 分布在相同位置，並且 actin會持續沿著層狀偽足呈現輻射狀的聚合。當 AQP3 受到抑制而極化時，actin 呈現平行且不具方向性聚合，使得細胞延展為細長紡錘狀進而抑制細胞遷移。在電
性量測試驗(electric cell-substrate impedance sensing, ECIS)發現，AQP3 會調控細胞層狀偽足的形成，改變細胞形態，進而影響細胞遷移。另外，可藉由阻抗值變化（± 50 ohm）偵測到 AQP3 調控細胞遷移時前進和後退的現象，以及抑制 AQP3時造成阻抗值變化量明顯降低，表示 AQP3 調控細胞形態進而影響細胞遷移、延展和貼附之情形。
綜合以上結果，本研究推測並且建立傷口癒合之細胞遷移模型。當傷口形成時，首先 AQP3 誘發細胞形成層狀偽足，吸引 actin monomers 聚集在 APQ3 的位置，然後層狀偽足會持續延展，藉由 actin monomers 會形成 actin filaments 維持層狀偽足的構形，促進細胞遷移。經由電性量測的方式可即時偵測到 AQP3 調控此傷口癒合之過程。

Aquaporins (AQPs)are integral membrane proteins transporting water or neutral partials in cells. AQPs have been demonstrated that facilitate epidermal cells migration and proliferation during the process of wound healing.AQPs generate wound healing via polarization and osmolarity change to regulate the distribution and remodeling of actin. This study was to observe the cell migration and adhesion regulated by AQP3 and actin, and to investigate how AQP3 conferred on the phenomenon of polarization for actin location and cell remolding in wound healing in order to establish the model of cell migration through AQP3 and actin.
The result of traditional wound healing assay showed that AQP3 was inhibited by nickel chloride (NiCl2)resulting in impaired cell migration and wound healing. The
polymerization of AQP3 and actin were inhibited in accompanied with no lamellipodium generation. It indicated that the polymerization of AQP3 and actin mediated lamellipodium generation to facilitate cell migration during wound healing. The immunofluorescence assay explored that AQP3 generally distributed around nuclei, and actin monomers colocalized with AQP3 distribution to polymerize radically along lamellipodium. Moreover, AQP3 deficiency interfered with polarization of AQP3, and caused the location of polymerized actin parallel and undirected. The assay of electric cell-substrate impedance sensing (ECIS) detected AQP3 mediated cell lamellipodium generation to affect cell migration and distribution resulting in impaired cell migration. Otherwise,the impedance change (± 50 ohm) was detectable in the forward and backward movement during cell migration, and the impedance was significantly decreased when AQP3 was repressed. It suggested that AQP3 mediated cell morphology for cell migration, extension and adhesion.
Regarding the findings of this study, it speculated and established a model of cell migration during wound healing. In the process of cell migration for wound healing, AQP3 induced cells to form lamellipodium and attract actin monomers to the location of AQP3 distribution, and lamellipodium further extended and actin filaments were formed from actin monomers to maintain the shape of lamellipodium. The ECIS assay can detect in real time that AQP3 regulated cell migration for wound healing.

參考文獻
1. Legrand, C., et al., Airway epithelial cell migration dynamics. MMP-9 role in cell-extracellular matrix remodeling. J Cell Biol, 1999. 146(2): p. 517-29.
2. Coppit, G., et al., The effects of mitomycin-C and stenting on airway wound healing after laryngotracheal reconstruction in a pig model. Int J Pediatr Otorhinolaryngol, 2000. 53(2): p. 125-35.
3. Papadopoulos, M.C., S. Saadoun, and A.S. Verkman, Aquaporins and cell migration. Pflugers Arch, 2008. 456(4): p. 693-700.
4. McCawley, L.J., P. O'Brien, and L.G. Hudson, Epidermal growth factor (EGF)- and scatter factor/hepatocyte growth factor (SF/HGF)- mediated keratinocyte migration is coincident with induction of matrix metalloproteinase (MMP)-9. J Cell Physiol, 1998. 176(2): p. 255-65.
5. Ji, L., J. Lim, and G. Danuser, Fluctuations of intracellular forces during cell protrusion. Nat Cell Biol, 2008. 10(12): p. 1393-400.
6. Hara-Chikuma, M. and A.S. Verkman, Aquaporin-3 facilitates epidermal cell migration and proliferation during wound healing. J Mol Med, 2008. 86(2): p. 221-31.
7. Baskovich, B., et al., Wound dressing components degrade proteins detrimental to wound healing. Int Wound J, 2008. 5(4): p. 543-51.
8. Barrientos, S., et al., Growth factors and cytokines in wound healing. Wound Repair Regen, 2008. 16(5): p. 585-601.
9. Schultz, G.S. and A. Wysocki, Interactions between extracellular matrix and growth factors in wound healing. Wound Repair Regen, 2009. 17(2): p. 153-62.
10. Vicente-Manzanares, M., D.J. Webb, and A.R. Horwitz, Cell migration at a glance. J Cell Sci, 2005. 118(Pt 21): p. 4917-9.
11. Herard, A.L., et al., Epithelial barrier integrity during in vitro wound repair of the airway epithelium. Am J Respir Cell Mol Biol, 1996. 15(5): p. 624-32.
12. Keenan, K.P., J.W. Combs, and E.M. McDowell, Regeneration of hamster tracheal epithelium after mechanical injury. I. Focal lesions: quantitative morphologic study of cell proliferation. Virchows Arch B Cell Pathol Incl Mol Pathol, 1982. 41(3): p. 193-214.
13. Etienne-Manneville, S., Polarity proteins in migration and invasion. Oncogene, 2008. 27(55): p. 6970-80.
14. Rorth, P., Collective cell migration. Annu Rev Cell Dev Biol, 2009. 25: p. 407-29.
15. Friedl, P. and D. Gilmour, Collective cell migration in morphogenesis, regeneration and cancer. Nat Rev Mol Cell Biol, 2009. 10(7): p. 445-57.
16. Horie, I., et al., Tumor necrosis factor-alpha decreases aquaporin-3 expression in DJM-1 keratinocytes. Biochem Biophys Res Commun, 2009. 387(3): p. 564-8.
17. Krane, C.M., et al., Altered regulation of aquaporin gene expression in allergen and IL-13-induced mouse models of asthma. Cytokine, 2009. 46(1): p. 111-8.
18. Loitto, V.M., T. Karlsson, and K.E. Magnusson, Water flux in cell motility: expanding the mechanisms of membrane protrusion. Cell Motil Cytoskeleton, 2009. 66(5): p. 237-47.
19. Ji, C., et al., Curcumin attenuates EGF-induced AQP3 up-regulation and cell migration in human ovarian cancer cells. Cancer Chemother Pharmacol, 2008. 62(5): p. 857-65.
20. Saadoun, S., et al., Impairment of angiogenesis and cell migration by targeted aquaporin-1 gene disruption. Nature, 2005. 434(7034): p. 786-92.
21. Verkman, A.S., More than just water channels: unexpected cellular roles of aquaporins. J Cell Sci, 2005. 118(Pt 15): p. 3225-32.
22. Diez, S., et al., Subsecond reorganization of the actin network in cell motility and chemotaxis. Proc Natl Acad Sci U S A, 2005. 102(21): p. 7601-6.
23. Small, J.V., et al., The lamellipodium: where motility begins. Trends Cell Biol, 2002. 12(3): p. 112-20.
24. Pollard, T.D. and G.G. Borisy, Cellular motility driven by assembly and disassembly of actin filaments. Cell, 2003. 112(4): p. 453-65.
25. Gerisch, G. and H.U. Keller, Chemotactic reorientation of granulocytes stimulated with micropipettes containing fMet-Leu-Phe. J Cell Sci, 1981. 52: p. 1-10.
26. Gerisch, G., et al., Chemoattractant-controlled accumulation of coronin at the leading edge of Dictyostelium cells monitored using a green fluorescent protein-coronin fusion protein. Curr Biol, 1995. 5(11): p. 1280-5.
27. Shaw, T.J. and P. Martin, Wound repair at a glance. J Cell Sci, 2009. 122(Pt 18): p. 3209-13.
28. Zelenina, M., et al., Copper inhibits the water and glycerol permeability of aquaporin-3. J Biol Chem, 2004. 279(50): p. 51939-43.
29. Zelenina, M., et al., Nickel and extracellular acidification inhibit the water permeability of human aquaporin-3 in lung epithelial cells. J Biol Chem, 2003. 278(32): p. 30037-43.
30. Ilina, O. and P. Friedl, Mechanisms of collective cell migration at a glance. J Cell Sci, 2009. 122(Pt 18): p. 3203-8.
31. Ridley, A.J., et al., Cell migration: integrating signals from front to back. Science, 2003. 302(5651): p. 1704-9.
32. Van Haastert, P.J. and P.N. Devreotes, Chemotaxis: signalling the way forward. Nat Rev Mol Cell Biol, 2004. 5(8): p. 626-34.
33. Kozuka, J., et al., Dynamic polymorphism of actin as activation mechanism for cell motility. Biosystems, 2007. 88(3): p. 273-82.
34. Fisher, C.I. and S.C. Kuo, Filament rigidity causes F-actin depletion from nonbinding surfaces. Proc Natl Acad Sci U S A, 2009. 106(1): p. 133-8.
35. Kruse, E., N. Uehlein, and R. Kaldenhoff, The aquaporins. Genome Biol, 2006. 7(2): p. 206.
36. Rash, J.E., et al., Direct immunogold labeling of aquaporin-4 in square arrays of astrocyte and ependymocyte plasma membranes in rat brain and spinal cord. Proc Natl Acad Sci U S A, 1998. 95(20): p. 11981-6.
37. Sui, H., et al., Structural basis of water-specific transport through the AQP1 water channel. Nature, 2001. 414(6866): p. 872-8.
38. Harries, W.E., et al., The channel architecture of aquaporin 0 at a 2.2-A resolution. Proc Natl Acad Sci U S A, 2004. 101(39): p. 14045-50.
39. Ma, T., et al., Defective secretion of saliva in transgenic mice lacking aquaporin-5 water channels. J Biol Chem, 1999. 274(29): p. 20071-4.
40. Gresz, V., et al., Immunolocalization of AQP-5 in rat parotid and submandibular salivary glands after stimulation or inhibition of secretion in vivo. Am J Physiol Gastrointest Liver Physiol, 2004. 287(1): p. G151-61.
41. Verkman, A.S., Applications of aquaporin inhibitors. Drug News Perspect, 2001. 14(7): p. 412-20.
42. Castle, N.A., Aquaporins as targets for drug discovery. Drug Discov Today, 2005. 10(7): p. 485-93.
43. Ma, T., et al., Severely impaired urinary concentrating ability in transgenic mice lacking aquaporin-1 water channels. J Biol Chem, 1998. 273(8): p. 4296-9.
44. Papadopoulos, M.C. and A.S. Verkman, Aquaporin-4 gene disruption in mice reduces brain swelling and mortality in pneumococcal meningitis. J Biol Chem, 2005. 280(14): p. 13906-12.
45. Binder, D.K., et al., Increased seizure threshold in mice lacking aquaporin-4 water channels. Neuroreport, 2004. 15(2): p. 259-62.
46. Da, T. and A.S. Verkman, Aquaporin-4 gene disruption in mice protects against impaired retinal function and cell death after ischemia. Invest Ophthalmol Vis Sci, 2004. 45(12): p. 4477-83.
47. Saadoun, S., et al., Involvement of aquaporin-4 in astroglial cell migration and glial scar formation. J Cell Sci, 2005. 118(Pt 24): p. 5691-8.
48. Solenov, E., et al., Sevenfold-reduced osmotic water permeability in primary astrocyte cultures from AQP-4-deficient mice, measured by a fluorescence quenching method. Am J Physiol Cell Physiol, 2004. 286(2): p. C426-32.
49. Jaeger, M., et al., The osmotic migration of cells in a solute gradient. Biophys J, 1999. 77(3): p. 1257-67.
50. Lim, J.H., et al., Extracellular signal-regulated kinase involvement in human astrocyte migration. Brain Res, 2007. 1164: p. 1-13.
51. Levin, M.H. and A.S. Verkman, Aquaporin-3-dependent cell migration and proliferation during corneal re-epithelialization. Invest Ophthalmol Vis Sci, 2006. 47(10): p. 4365-72.
52. Thiagarajah, J.R., D. Zhao, and A.S. Verkman, Impaired enterocyte proliferation in aquaporin-3 deficiency in mouse models of colitis. Gut, 2007. 56(11): p. 1529-35.
53. Crane, J.M. and A.S. Verkman, Long-range nonanomalous diffusion of quantum dot-labeled aquaporin-1 water channels in the cell plasma membrane. Biophys J, 2008. 94(2): p. 702-13.
54. Nielsen, S., et al., Specialized membrane domains for water transport in glial cells: high-resolution immunogold cytochemistry of aquaporin-4 in rat brain. J Neurosci, 1997. 17(1): p. 171-80.
55. Disanza, A., et al., Actin polymerization machinery: the finish line of signaling networks, the starting point of cellular movement. Cell Mol Life Sci, 2005. 62(9): p. 955-70.
56. Schwab, A., et al., Cells move when ions and water flow. Pflugers Arch, 2007. 453(4): p. 421-32.
57. Dong, C., S. Aznavoorian, and L.A. Liotta, Two phases of pseudopod protrusion in tumor cells revealed by a micropipette. Microvasc Res, 1994. 47(1): p. 55-67.
58. Rabinovitch, M. and M.J. DeStefano, Spontaneous migration of normal human polymorphonuclear neutrophils under agarose: enhancement by media of lowered pH or osmolality. J Reticuloendothel Soc, 1981. 29(5): p. 329-39.
59. Charras, G.T., et al., Non-equilibration of hydrostatic pressure in blebbing cells. Nature, 2005. 435(7040): p. 365-9.
60. Barber, M.A. and H.C. Welch, PI3K and RAC signalling in leukocyte and cancer cell migration. Bull Cancer, 2006. 93(5): p. E44-52.
61. Lopez, J.I., J.K. Mouw, and V.M. Weaver, Biomechanical regulation of cell orientation and fate. Oncogene, 2008. 27(55): p. 6981-93.
62. Nascimento, C.F., et al., Role of MMP9 on invadopodia formation in cells from adenoid cystic carcinoma. Study by laser scanning confocal microscopy. Microsc Res Tech, 2009.
63. Ashkin, A., Optical trapping and manipulation of neutral particles using lasers. Proc Natl Acad Sci U S A, 1997. 94(10): p. 4853-60.
64. Nemet, B.A. and M. Cronin-Golomb, Measuring microscopic viscosity with optical tweezers as a confocal probe. Appl Opt, 2003. 42(10): p. 1820-32.
65. Dormann, D. and C.J. Weijer, Imaging of cell migration. EMBO J, 2006. 25(15): p. 3480-93.
66. Wegener, J., C.R. Keese, and I. Giaever, Electric cell-substrate impedance sensing (ECIS) as a noninvasive means to monitor the kinetics of cell spreading to artificial surfaces. Exp Cell Res, 2000. 259(1): p. 158-66.
67. Keese, C.R., et al., Electrical wound-healing assay for cells in vitro. Proc Natl Acad Sci U S A, 2004. 101(6): p. 1554-9.
68. Zhao, M., Electrical fields in wound healing-An overriding signal that directs cell migration. Semin Cell Dev Biol, 2009. 20(6): p. 674-82.
69. Earley, S. and G.E. Plopper, Disruption of focal adhesion kinase slows transendothelial migration of AU-565 breast cancer cells. Biochem Biophys Res Commun, 2006. 350(2): p. 405-12.
70. Gorshkova, I., et al., Protein kinase C-epsilon regulates sphingosine 1-phosphate-mediated migration of human lung endothelial cells through activation of phospholipase D2, protein kinase C-zeta, and Rac1. J Biol Chem, 2008. 283(17): p. 11794-806.
71. Balasubramanian, L., et al., Impedance analysis of renal vascular smooth muscle cells. Am J Physiol Cell Physiol, 2008. 295(4): p. C954-65.
72. Cao, C., et al., EGFR-mediated expression of aquaporin-3 is involved in human skin fibroblast migration. Biochem J, 2006. 400(2): p. 225-34.
73. Zelenina, M., et al., Water permeability of aquaporin-4 is decreased by protein kinase C and dopamine. Am J Physiol Renal Physiol, 2002. 283(2): p. F309-18.
74. Giaever, I. and C.R. Keese, Micromotion of mammalian cells measured electrically. Proc Natl Acad Sci U S A, 1991. 88(17): p. 7896-900.
75. Tiruppathi, C., et al., Electrical method for detection of endothelial cell shape change in real time: assessment of endothelial barrier function. Proc Natl Acad Sci U S A, 1992. 89(17): p. 7919-23.
76. Nikula, K.J., et al., The response of the rat tracheal epithelium to ozone exposure. Injury, adaptation, and repair. Am J Pathol, 1988. 131(2): p. 373-84.
77. Erjefalt, J.S., et al., In vivo restitution of airway epithelium. Cell Tissue Res, 1995. 281(2): p. 305-16.
78. Mattila, P.K. and P. Lappalainen, Filopodia: molecular architecture and cellular functions. Nat Rev Mol Cell Biol, 2008. 9(6): p. 446-54.
79. Nejsum, L.N. and W.J. Nelson, A molecular mechanism directly linking E-cadherin adhesion to initiation of epithelial cell surface polarity. J Cell Biol, 2007. 178(2): p. 323-35.
80. Mark, S., et al., Physical model of the dynamic instability in an expanding cell culture. Biophys J, 2010. 98(3): p. 361-70.
81. Konishi, H., et al., Latrunculin a has a strong anticancer effect in a peritoneal dissemination model of human gastric cancer in mice. Anticancer Res, 2009. 29(6): p. 2091-7.
82. 陳宣毅，細胞爬行的物理學。 物理雙月刊，2007，29 (6): 1029-1033.