幾丁聚醣(chitosan)因具有將上皮細胞間tight junction (TJ)打開的特性，而被應用於口服或鼻腔給藥的藥物吸收促進劑，然而幾丁聚醣打開細胞間TJ的詳細機制尚未清楚了解。因此本研究分為兩個方向進行探討：第一部分為幾丁聚醣與細胞膜蛋白間的作用，首先以抗體阻斷膜蛋白與幾丁聚醣間可能的結合後，加入幾丁聚醣，利用transepithelial electrical resistance (TEER)測量TJ打開的情形，結果顯示coxsackievirus and adnovirus receptor (CAR)被抗體阻斷後，會抑制幾丁聚醣打開TJ的能力，因此進一步利用shRNA抑制細胞中CAR的表現量，以確認CAR在幾丁聚醣打開TJ中所扮演的角色。第二部分為利用microarray篩選出經幾丁聚醣處理後細胞所產生的基因表現變化情形，然後以RT-PCR測定以幾丁聚醣處理不移除及移除使TJ回復的兩組細胞中六種基因的表現變化。結果顯示TJ穿膜蛋白claudin-4在幾丁聚醣移除後1小時，基因表現會增為原先的6倍，而蛋白質表現量也會在1~4小時有2倍的增加。而在幾丁聚醣打開TJ的過程中，也經由分離細胞質/膜蛋白及免疫螢光染色的方式發現，隨著幾丁聚醣處理的時間，細胞膜內的claudin-4會有梯度性的下降情形。本實驗初步驗證幾丁聚醣打開TJ及其移除後TJ的回復過程中，claudin-4的表現量會受到顯著的影響。

摘要 I
誌謝 II
目錄 III
第一章 序論 1
1.1 幾丁聚醣 1
1.2 Tight Junction 2
1.2.1 Tight Junction功能 2
1.2.1 Tight Junction組成 3
1.3 Tight junction穿膜蛋白 4
1.3.1 Junctional adhesion molecule (JAM) 4
1.3.2 Coxsackievirus and Adnovirus Receptor (CAR) 5
1.3.3 Claudin 7
1.4 GPI-anchored Protein Decay-accelerating Factor (DAF) 9
1.5 訊息傳遞路徑 10
1.5.1 Protein Kinase C (PKC) 10
1.5.2 Rho Family GTPases 12
1.6 研究動機與目的 13
第二章 細胞表面膜蛋白與幾丁聚醣打開TJ的關聯性 15
2.1 研究目的 15
2.2 材料與方法 15
2.2.1細胞培養 15
2.2.1.1 Caco-2細胞 15
2.2.1.2配置SMEM培養液 16
2.2.1.3 配置Hank’s buffer (HBSS) 16
2.2.1.4配置Caco-2細胞培養液 16
2.2.1.5 細胞繼代 17
2.2.2 Tight junction通透度測試 17
2.2.2.1 培養Caco-2單層細胞 17
2.2.2.2 Transepithelial electrical resistance (TEER)測定 18
2.2.2.3配置幾丁聚醣溶液 19
2.2.2.4 抗體阻斷 19
2.2.3 蛋白質的表現與抑制 20
2.2.3.1 抑制CAR表現量的Caco-2細胞 20
2.2.3.2 免疫螢光染色 20
2.2.4基因表現量 21
2.2.4.1 RNA萃取 21
2.2.4.2 反轉錄反應 22
2.2.4.3 Real-time PCR 23
2.3 實驗結果 24
2.3.1 單純抗體對TEER的影響 24
2.3.2 抗體阻斷TEER 25
2.3.3 電腦模擬 26
2.3.4 抑制CAR蛋白質表現 27
2.3.5 抑制CAR表現細胞TEER 29
2.4 結果討論 30
第三章 幾丁聚醣造成的基因與蛋白質表現變化 32
3.1研究目的 32
3.2材料與方法 32
3.2.1基因表現 32
3.2.1.1 RNA萃取 32
3.2.1.2 Microarray Assay 32
3.2.1.3 反轉錄反應 33
3.2.1.4 Real-time PCR 33
3.2.2 蛋白質表現 33
3.2.2.1 蛋白質萃取 33
3.2.2.2 西方墨點法 (Western Blot) 34
3.2.2.3 免疫螢光染色 35
3.3 實驗結果 35
3.3.1 Microarray 35
3.3.2 幾丁聚醣刺激造成的基因表現變化 36
3.3.3 幾丁聚醣刺激後移除claudin蛋白質表現變化 39
3.3.4 幾丁聚醣刺激造成claudin-4蛋白質表現變化 42
3.4 結果討論 44
文獻參考 46

1.Illum, L., N.F. Farraj, and S.S. Davis, Chitosan as a novel nasal delivery system for peptide drugs. Pharm Res, 1994. 11(8): p. 1186-9.
2.Thanou, M., J.C. Verhoef, and H.E. Junginger, Chitosan and its derivatives as intestinal absorption enhancers. Adv Drug Deliv Rev, 2001. 50 Suppl 1: p. S91-101.
3.Smith, J., E. Wood, and M. Dornish, Effect of chitosan on epithelial cell tight junctions. Pharm Res, 2004. 21(1): p. 43-9.
4.Artursson, P., et al., Effect of chitosan on the permeability of monolayers of intestinal epithelial cells (Caco-2). Pharm Res, 1994. 11(9): p. 1358-61.
5.Smith, J.M., M. Dornish, and E.J. Wood, Involvement of protein kinase C in chitosan glutamate-mediated tight junction disruption. Biomaterials, 2005. 26(16): p. 3269-76.
6.Tsukita, S., M. Furuse, and M. Itoh, Multifunctional strands in tight junctions. Nat Rev Mol Cell Biol, 2001. 2(4): p. 285-93.
7.Hossain, Z. and T. Hirata, Molecular mechanism of intestinal permeability: interaction at tight junctions. Mol Biosyst, 2008. 4(12): p. 1181-5.
8.Matter, K. and M.S. Balda, Signalling to and from tight junctions. Nat Rev Mol Cell Biol, 2003. 4(3): p. 225-36.
9.Coyne, C.B. and J.M. Bergelson, CAR: a virus receptor within the tight junction. Adv Drug Deliv Rev, 2005. 57(6): p. 869-82.
10.Van Itallie, C.M., et al., ZO-1 stabilizes the tight junction solute barrier through coupling to the perijunctional cytoskeleton. Mol Biol Cell, 2009. 20(17): p. 3930-40.
11.Nagumo, Y., et al., Cofilin mediates tight-junction opening by redistributing actin and tight-junction proteins. Biochem Biophys Res Commun, 2008. 377(3): p. 921-5.
12.Stamatovic, S.M., et al., Potential role of MCP-1 in endothelial cell tight junction 'opening': signaling via Rho and Rho kinase. J Cell Sci, 2003. 116(Pt 22): p. 4615-28.
13.Martin-Padura, I., et al., Junctional adhesion molecule, a novel member of the immunoglobulin superfamily that distributes at intercellular junctions and modulates monocyte transmigration. J Cell Biol, 1998. 142(1): p. 117-27.
14.Johnson-Leger, C.A., et al., Junctional adhesion molecule-2 (JAM-2) promotes lymphocyte transendothelial migration. Blood, 2002. 100(7): p. 2479-86.
15.Ebnet, K., et al., Junctional adhesion molecule interacts with the PDZ domain-containing proteins AF-6 and ZO-1. J Biol Chem, 2000. 275(36): p. 27979-88.
16.Ebnet, K., et al., The cell polarity protein ASIP/PAR-3 directly associates with junctional adhesion molecule (JAM). EMBO J, 2001. 20(14): p. 3738-48.
17.Itoh, M., et al., Junctional adhesion molecule (JAM) binds to PAR-3: a possible mechanism for the recruitment of PAR-3 to tight junctions. J Cell Biol, 2001. 154(3): p. 491-7.
18.Liu, Y., et al., Human junction adhesion molecule regulates tight junction resealing in epithelia. J Cell Sci, 2000. 113 ( Pt 13): p. 2363-74.
19.Barton, E.S., et al., Junction adhesion molecule is a receptor for reovirus. Cell, 2001. 104(3): p. 441-51.
20.Tyler, K.L., et al., Reoviruses and the host cell. Trends Microbiol, 2001. 9(11): p. 560-4.
21.Bergelson, J.M., et al., Isolation of a common receptor for Coxsackie B viruses and adenoviruses 2 and 5. Science, 1997. 275(5304): p. 1320-3.
22.Tomko, R.P., R. Xu, and L. Philipson, HCAR and MCAR: the human and mouse cellular receptors for subgroup C adenoviruses and group B coxsackieviruses. Proc Natl Acad Sci U S A, 1997. 94(7): p. 3352-6.
23.Cohen, C.J., et al., The coxsackievirus and adenovirus receptor is a transmembrane component of the tight junction. Proc Natl Acad Sci U S A, 2001. 98(26): p. 15191-6.
24.Walters, R.W., et al., Adenovirus fiber disrupts CAR-mediated intercellular adhesion allowing virus escape. Cell, 2002. 110(6): p. 789-99.
25.Raschperger, E., et al., The coxsackie- and adenovirus receptor (CAR) is an in vivo marker for epithelial tight junctions, with a potential role in regulating permeability and tissue homeostasis. Exp Cell Res, 2006. 312(9): p. 1566-80.
26.Zen, K., et al., Neutrophil migration across tight junctions is mediated by adhesive interactions between epithelial coxsackie and adenovirus receptor and a junctional adhesion molecule-like protein on neutrophils. Mol Biol Cell, 2005. 16(6): p. 2694-703.
27.Schneeberger, E.E. and R.D. Lynch, The tight junction: a multifunctional complex. Am J Physiol Cell Physiol, 2004. 286(6): p. C1213-28.
28.Brennan, K., et al., Tight junctions: a barrier to the initiation and progression of breast cancer? J Biomed Biotechnol. 2010: p. 460607.
29.Turksen, K. and T.C. Troy, Barriers built on claudins. J Cell Sci, 2004. 117(Pt 12): p. 2435-47.
30.Hartsock, A. and W.J. Nelson, Adherens and tight junctions: structure, function and connections to the actin cytoskeleton. Biochim Biophys Acta, 2008. 1778(3): p. 660-9.
31.Furuse, M., et al., Claudin-based tight junctions are crucial for the mammalian epidermal barrier: a lesson from claudin-1-deficient mice. J Cell Biol, 2002. 156(6): p. 1099-111.
32.Furuse, M., et al., Conversion of zonulae occludentes from tight to leaky strand type by introducing claudin-2 into Madin-Darby canine kidney I cells. J Cell Biol, 2001. 153(2): p. 263-72.
33.Banan, A., et al., theta Isoform of protein kinase C alters barrier function in intestinal epithelium through modulation of distinct claudin isotypes: a novel mechanism for regulation of permeability. J Pharmacol Exp Ther, 2005. 313(3): p. 962-82.
34.Leotlela, P.D., et al., Claudin-1 overexpression in melanoma is regulated by PKC and contributes to melanoma cell motility. Oncogene, 2007. 26(26): p. 3846-56.
35.Reid, K.B. and A.J. Day, Structure-function relationships of the complement components. Immunol Today, 1989. 10(6): p. 177-80.
36.Coyne, C.B. and J.M. Bergelson, Virus-induced Abl and Fyn kinase signals permit coxsackievirus entry through epithelial tight junctions. Cell, 2006. 124(1): p. 119-31.
37.Bergelson, J.M., et al., Clinical coxsackievirus B isolates differ from laboratory strains in their interaction with two cell surface receptors. J Infect Dis, 1997. 175(3): p. 697-700.
38.Shafren, D.R., D.T. Williams, and R.D. Barry, A decay-accelerating factor-binding strain of coxsackievirus B3 requires the coxsackievirus-adenovirus receptor protein to mediate lytic infection of rhabdomyosarcoma cells. J Virol, 1997. 71(12): p. 9844-8.
39.Martino, T.A., et al., Cardiovirulent coxsackieviruses and the decay-accelerating factor (CD55) receptor. Virology, 1998. 244(2): p. 302-14.
40.Mellor, H. and P.J. Parker, The extended protein kinase C superfamily. Biochem J, 1998. 332 ( Pt 2): p. 281-92.
41.Citi, S., et al., Cytoskeletal involvement in the modulation of cell-cell junctions by the protein kinase inhibitor H-7. J Cell Sci, 1994. 107 ( Pt 3): p. 683-92.
42.Lin, D., et al., A mammalian PAR-3-PAR-6 complex implicated in Cdc42/Rac1 and aPKC signalling and cell polarity. Nat Cell Biol, 2000. 2(8): p. 540-7.
43.Joberty, G., et al., The cell-polarity protein Par6 links Par3 and atypical protein kinase C to Cdc42. Nat Cell Biol, 2000. 2(8): p. 531-9.
44.Suzuki, A., et al., Atypical protein kinase C is involved in the evolutionarily conserved par protein complex and plays a critical role in establishing epithelia-specific junctional structures. J Cell Biol, 2001. 152(6): p. 1183-96.
45.Kroschewski, R., A. Hall, and I. Mellman, Cdc42 controls secretory and endocytic transport to the basolateral plasma membrane of MDCK cells. Nat Cell Biol, 1999. 1(1): p. 8-13.
46.Nunbhakdi-Craig, V., et al., Protein phosphatase 2A associates with and regulates atypical PKC and the epithelial tight junction complex. J Cell Biol, 2002. 158(5): p. 967-78.
47.Balda, M.S., et al., Assembly and sealing of tight junctions: possible participation of G-proteins, phospholipase C, protein kinase C and calmodulin. J Membr Biol, 1991. 122(3): p. 193-202.
48.Nusrat, A., et al., Rho protein regulates tight junctions and perijunctional actin organization in polarized epithelia. Proc Natl Acad Sci U S A, 1995. 92(23): p. 10629-33.
49.Zhong, C., M.S. Kinch, and K. Burridge, Rho-stimulated contractility contributes to the fibroblastic phenotype of Ras-transformed epithelial cells. Mol Biol Cell, 1997. 8(11): p. 2329-44.
50.Hasegawa, H., et al., Opposite regulation of transepithelial electrical resistance and paracellular permeability by Rho in Madin-Darby canine kidney cells. J Biol Chem, 1999. 274(30): p. 20982-8.
51.Jou, T.S., E.E. Schneeberger, and W.J. Nelson, Structural and functional regulation of tight junctions by RhoA and Rac1 small GTPases. J Cell Biol, 1998. 142(1): p. 101-15.
52.Wojciak-Stothard, B., et al., Rho and Rac but not Cdc42 regulate endothelial cell permeability. J Cell Sci, 2001. 114(Pt 7): p. 1343-55.
53.Braga, V.M., et al., Regulation of cadherin function by Rho and Rac: modulation by junction maturation and cellular context. Mol Biol Cell, 1999. 10(1): p. 9-22.
54.Adamson, P., et al., Lymphocyte migration through brain endothelial cell monolayers involves signaling through endothelial ICAM-1 via a rho-dependent pathway. J Immunol, 1999. 162(5): p. 2964-73.
55.Fujita, H., et al., Molecular decipherment of Rho effector pathways regulating tight-junction permeability. Biochem J, 2000. 346 Pt 3: p. 617-22.
56.Hirase, T., et al., Regulation of tight junction permeability and occludin phosphorylation by Rhoa-p160ROCK-dependent and -independent mechanisms. J Biol Chem, 2001. 276(13): p. 10423-31.
57.Etienne-Manneville, S. and A. Hall, Rho GTPases in cell biology. Nature, 2002. 420(6916): p. 629-35.
58.Hecht, G., et al., Expression of the catalytic domain of myosin light chain kinase increases paracellular permeability. Am J Physiol, 1996. 271(5 Pt 1): p. C1678-84.
59.Turner, J.R., et al., Physiological regulation of epithelial tight junctions is associated with myosin light-chain phosphorylation. Am J Physiol, 1997. 273(4 Pt 1): p. C1378-85.
60.Lanaspa, M.A., et al., Hypertonic stress increases claudin-4 expression and tight junction integrity in association with MUPP1 in IMCD3 cells. Proc Natl Acad Sci U S A, 2008. 105(41): p. 15797-802.