臺灣博碩士論文加值系統

基底膜(basement membrane, BM)是由第四型膠原蛋白(type IV collagen)、層黏蛋白(laminin)及蛋白多醣(proteoglycan)等物質所構成，主要功能是扮演維持上皮及內皮細胞型態與細胞附著及做為細胞生長支架(scaffold)的功能。硫酸肝素蛋白多醣(HSPG)為BM中重要的蛋白多醣類。先前研究指出HSPG具有促進神經纖維生長的功能，而珍珠素(perlecan)即屬於HSPG的一種，本研究是在探討基底膜中硫酸肝素蛋白多醣(heparan sulfate proteoglycan)珍珠素(perlecan)在神經系統所扮演的角色與功能，先前的研究中證實重組的人類珍珠素蛋白多醣第五片段(human perlecan domain V; H.Pln DV)在以laminin為基材貼覆的環境下具有促進背根神經節神經纖維生長的能力，因此本實驗主要目的是以重組基因技術(recombinant DNA technology)將perlecan domain V分成兩個含類似組成的片段(H.Pln DV-I, H.Pln DV-II)探討其對神經纖維生長之影響及作用機轉，研究結果顯示兩片段都會促進神經纖維的生長，先前文獻中顯示perlecan會與乙醯膽鹼酯酵素(AchE)有交互作用且AchE有促進神經纖維生長之作用，本研究第二部分探討perlecan與AchE的作用是否是透過perlecan domain V。結果顯示H.Pln DV, DV-I, DV-II可直接與AchE結合。推測H.Pln DV可能藉由與AchE的結合促進神經纖維的生長的。

The major components of basement membrane (BM) are type IV collagen, laminin and proteoglycans. The basement membrane fuctions as a scaffold to maintain the morphology of epithelium and endothelial cells. The major type of proteoglycan in BM is heparan sulfate proteoglycan (HSPG). Previous study has shown that HSPG can promote the neurite outgrowth. Our studies are to understand the role of perlecan, which is the predominant HSPG in BM in the neurite outgrowth. Our previous study showed that recomibnant human perlecan domain V could promote the neurite outgrowth of dorsal root ganglion (DRG) neurons in laminin coating. Here we divided the perlecan domain V (H.Pln DV) into two smaller fragment to identify the minimum fragment required for promoting the neurite outgrowth. The result showed that both of these two smaller fragments derived from perlecan domain V could promote the neurite outgrowth of DRG neurons. Previously study showed that perlecan and acetylcholinesterase (AchE) could interact with each other and AchE could promote the neurite outgrowth. Our further studies showed that H.Pln DV, H.Pln DV-I, H.Pln DV-II could bind to AchE. It suggests that H.Pln DV promoting the neurite outgrowth may be mediated by binding to AchE.

目錄
目錄.............................................................................................................I
圖表目錄…..............................................................................…………IV
附錄目錄….......................................................................…….……..…VI
縮寫表........................................................................................................1
中文摘要....................................................................................................2
英文摘要....................................................................................................3
第一章 文獻探討........................................................................................4
第二章 實驗目的......................................................................................13
第三章 實驗設計......................................................................................14
實驗一、聚合酵素連鎖反應(polymerase chain reaction, PCR)增幅人類珍珠素蛋白第五片段片段一以及片段二………………………..14
實驗二、重組Human perlecan domain V-I、Human perlecan domain V-II質體DNA…..................................................................................16
實驗三、Human perlecan domain V-I, Human perlecan domain V-II蛋白質表現..............................................................................................19
實驗四、Human perlecan domain V-I, Human perlecan domain V-II蛋白質純化..............................................................................................21
實驗五、西方點墨法(Western blot analysis).......................................25
實驗六、大鼠大腦組織萃取珍珠素(Perlecan-R)之純化及定性分析
蛋白多醣(Proteoglycan)之萃取純化…..............................................29
實驗七、初代背根神經節神經細胞培養............................................32
實驗八、背根神經節神經細胞神經纖維生長實驗及分析…............33
實驗九、背根神經節神經細胞螢光免液染色....................................37
實驗十、乙醯膽鹼酯酵素活性試驗(AchE activity assay)……..........38
實驗十一、乙醯膽鹼酯酵素(AchE)接和試驗 (binding-assay)….... 39
實驗十二、perlecan domain V(H.Pln DV)與laminin(LN)對乙醯膽鹼酯酵素(acetylcholinesterase, AchE)之競爭性結合試驗....................41
實驗十三、免疫沉澱法((Immunoprecipitation, IP) ............................42
實驗十四、統計分析方法........................ ...........................................45
第四章 實驗結果......................................................................................46
1. Human perlecan domain V-I，domain V-II之選殖…..................46
2. Human perlecan domain V-I、Human perlecan domain V-II 重組蛋白之表現….............................................................................48
3. Human perlecan domain V-I、Human perlecan domain V-II 重組蛋白之純化….............................................................................49
4. 大鼠大腦萃取之蛋白多醣圓點點墨法(Dot blot)之定性.........50
5. Human perlecan domain V-I及Human perlecan domain V-II對於背根神經節神經細胞神經纖維生長之影響.............................51
6. DRG神經細胞中之乙醯膽鹼酯酵素(AchE)在不同基質活性之影響….......................................................................…………..54
7. PC12細胞中之乙醯膽鹼酯酵素(AchE)與不同基質之間之結和能力.......................................................................………..……55
8. perlecan domain V(H.Pln DV)與laminin(LN)對乙醯膽鹼酯酵素(acetylcholinesterase, AchE)之競爭性結合試驗…...............56
9. 乙醯膽鹼酯酵素與Perlecan domain之免疫沉澱實驗….........57
第五章 討論…......................................................................……………58
第六章 結論…..............................................................................………63
第七章 未來研究方向..............................................................................64
第八章 參考文獻......................................................................................65
第九章 實驗圖表......................................................................................69
第十章 附錄..............................................................................................97

1.Kallunki, P. & Tryggvason, K. Human basement membrane heparan sulfate proteoglycan core protein: a 467-kD protein containing multiple domains resembling elements of the low density lipoprotein receptor, laminin, neural cell adhesion molecules, and epidermal growth factor. J Cell Biol 116, 559-571 (1992).
2.Timpl, R. Macromolecular organization of basement membranes. Curr Opin Cell Biol 8, 618-624 (1996).
3.Shee, W.L., Ong, W.Y. & Lim, T.M. Distribution of perlecan in mouse hippocampus following intracerebroventricular kainate injections. Brain Res 799, 292-300 (1998).
4.Knox, S.M. & Whitelock, J.M. Perlecan: how does one molecule do so many things? Cell Mol Life Sci 63, 2435-2445 (2006).
5.Handler, M., Yurchenco, P.D. & Iozzo, R.V. Developmental expression of perlecan during murine embryogenesis. Dev Dyn 210, 130-145 (1997).
6.Iozzo, R.V. Matrix proteoglycans: from molecular design to cellular function. Annu Rev Biochem 67, 609-652 (1998).
7.Iozzo, R.V. Basement membrane proteoglycans: from cellar to ceiling. Nat Rev Mol Cell Biol 6, 646-656 (2005).
8.French, M.M., et al. Chondrogenic activity of the heparan sulfate proteoglycan perlecan maps to the N-terminal domain I. J Bone Miner Res 17, 48-55 (2002).
9.Yang, W., et al. Chondrogenic differentiation on perlecan domain I, collagen II, and bone morphogenetic protein-2-based matrices. Tissue Eng 12, 2009-2024 (2006).
10.Conde-Knape, K. Heparan sulfate proteoglycans in experimental models of diabetes: a role for perlecan in diabetes complications. Diabetes Metab Res Rev 17, 412-421 (2001).
11.Forsberg, E. & Kjellen, L. Heparan sulfate: lessons from knockout mice. J Clin Invest 108, 175-180 (2001).
12.Smith, S.M., West, L.A. & Hassell, J.R. The core protein of growth plate perlecan binds FGF-18 and alters its mitogenic effect on chondrocytes. Arch Biochem Biophys 468, 244-251 (2007).
13.Balasubramani, M., Bier, M.E., Hummel, S., Schneider, W.J. & Halfter, W. Perlecan and its immunoglobulin like domain IV are abundant in vitreous and serum of the chick embryo. Matrix Biol 23, 143-152 (2004).
14.Arikawa-Hirasawa, E., Rossi, S.G., Rotundo, R.L. & Yamada, Y. Absence of acetylcholinesterase at the neuromuscular junctions of perlecan-null mice. Nat Neurosci 5, 119-123 (2002).
15.壽天德, 郭. 神經的損傷與再生. 神經生物學, 189-216 (2003).
16.Costell, M., et al. Perlecan maintains the integrity of cartilage and some basement membranes. J Cell Biol 147, 1109-1122 (1999).
17.Hartmann, U. & Maurer, P. Proteoglycans in the nervous system--the quest for functional roles in vivo. Matrix Biol 20, 23-35 (2001).
18.Braunewell, K.H., et al. Functional involvement of sciatic nerve-derived versican- and decorin-like molecules and other chondroitin sulphate proteoglycans in ECM-mediated cell adhesion and neurite outgrowth. Eur J Neurosci 7, 805-814 (1995).
19.Freire, E., Gomes, F.C., Linden, R., Neto, V.M. & Coelho-Sampaio, T. Structure of laminin substrate modulates cellular signaling for neuritogenesis. J Cell Sci 115, 4867-4876 (2002).
20.Skubitz, A.P., Letourneau, P.C., Wayner, E. & Furcht, L.T. Synthetic peptides from the carboxy-terminal globular domain of the A chain of laminin: their ability to promote cell adhesion and neurite outgrowth, and interact with heparin and the beta 1 integrin subunit. J Cell Biol 115, 1137-1148 (1991).
21.Whitelock, J.M. & Iozzo, R.V. Heparan sulfate: a complex polymer charged with biological activity. Chem Rev 105, 2745-2764 (2005).
22.Knox, S., Melrose, J. & Whitelock, J. Electrophoretic, biosensor, and bioactivity analyses of perlecans of different cellular origins. Proteomics 1, 1534-1541 (2001).
23.Gonzalez, E.M., Mongiat, M., Slater, S.J., Baffa, R. & Iozzo, R.V. A novel interaction between perlecan protein core and progranulin: potential effects on tumor growth. J Biol Chem 278, 38113-38116 (2003).
24.Mongiat, M., et al. Perlecan protein core interacts with extracellular matrix protein 1 (ECM1), a glycoprotein involved in bone formation and angiogenesis. J Biol Chem 278, 17491-17499 (2003).
25.Hossain, W.A. & Morest, D.K. Fibroblast growth factors (FGF-1, FGF-2) promote migration and neurite growth of mouse cochlear ganglion cells in vitro: immunohistochemistry and antibody perturbation. J Neurosci Res 62, 40-55 (2000).
26.Xie, Y., et al. The leukocyte common antigen-related protein tyrosine phosphatase receptor regulates regenerative neurite outgrowth in vivo. J Neurosci 21, 5130-5138 (2001).
27.Powell, S.K. & Kleinman, H.K. Neuronal laminins and their cellular receptors. Int J Biochem Cell Biol 29, 401-414 (1997).
28.Brown, J.C., Sasaki, T., Gohring, W., Yamada, Y. & Timpl, R. The C-terminal domain V of perlecan promotes beta1 integrin-mediated cell adhesion, binds heparin, nidogen and fibulin-2 and can be modified by glycosaminoglycans. Eur J Biochem 250, 39-46 (1997).
29.Hausmann, O.N. Post-traumatic inflammation following spinal cord injury. Spinal Cord 41, 369-378 (2003).
30.Lander, A.D., Fujii, D.K., Gospodarowicz, D. & Reichardt, L.F. Characterization of a factor that promotes neurite outgrowth: evidence linking activity to a heparan sulfate proteoglycan. J Cell Biol 94, 574-585 (1982).
31.Johnson, G., Swart, C. & Moore, S.W. Interaction of acetylcholinesterase with the G4 domain of the laminin alpha1-chain. Biochem J 411, 507-514 (2008).
32.Paraoanu, L.E. & Layer, P.G. Acetylcholinesterase in cell adhesion, neurite growth and network formation. FEBS J 275, 618-624 (2008).
33.Layer, P.G., Weikert, T. & Alber, R. Cholinesterases regulate neurite growth of chick nerve cells in vitro by means of a non-enzymatic mechanism. Cell Tissue Res 273, 219-226 (1993).