神經元的貼附和突觸生長在大腦發展中扮演關鍵的角色。然而神經元的生物力學和突觸之間的關係仍未被深入探討。突觸生長可藉由神經生長因子或是化學模擬低氧調控對PC12細胞株誘發進而模擬活體內的神經元發展過程。在本研究中，我們針對細胞型態的改變和貼附性質的關係探討。藉由細胞刮取來量化不同突觸誘發模型中貼附性質的改變。實驗中選擇以缺氧模擬藥劑(CoCl2)和神經生長因子(NGF)作用24小時來對PC12細胞株進行分化。
結果顯示CoCl2和NGF都會造成細胞型態上顯著的差異, 包括細胞貼附面積、突觸生長和真圓度的改變；兩種分化模型唯一的型態差異在於NGF分化下細胞貼附面積顯著大於以CoCl2分化。在細胞力學表現上，兩者皆可顯著提高細胞最大貼附力和外力做功；在去除細胞尺度因素之後，單位面積下的最大貼附力只在NGF模型中有顯著上升; 分析分化後的細胞極性對刮取的影響發現在兩組分化模型中, 刮取方向平行細胞主軸所需施加的外力和功皆高於正交方向; 計算相對的細胞勁度發現兩組分化模型有顯著差異且細胞勁度的量測與方向無關。螢光染色發現增加低氧誘導因子(hypoxia inducible factor-1α ,HIF-1α)表現, 進一步確定CoCl2分化模型中細胞進行低氧的調控。
細胞的外型和結構相關，實驗結果發現類似的分化形態仍然存在不同的力學性質。目前的細胞模型無法描述攤平且不規則外型的細胞貼附性質，藉由對於分化細胞進行極性的探討使我們對於刮取機構的特性有更深入的了解，且在未來能有更廣泛的應用。

The cell attachment and neurite outgrowth are critical in neuron development. However, the relationships between cellular biomechanical property and neural outgrowth are still poor understanding. The neurite outgrowth can be induced by growth factor or chemical reagents in PC12 cells to imitate the neuron development in vitro. In present study, we are interested in the relationship between the changes of cell morphology and mechanical remodeling of cell adhesiveness. We used cytodetachment to quantitatively measure the maximal cell adhesion force of different induction treatments. The inducing model was achieved by 24 hr treatment of hypoxia mimic chemical reagent (CoCl2) or nerve growth factor (NGF) on PC12 cells.
Our results showed that both the CoCl2 and NGF treatments caused significantly morphological changes including cell area, neurite ourtgrowth and cell roundness; the only difference was that the cell area of NGF group was significantly greater than that of CoCl2. Comparing the mechanical behavior, we found that both of the treatments increase maximal adhesion force and total work; after dividing the force to the cell area, the normalized force significantly increased in NGF group. The cell orientation will effect maximal adhesion force and work after cell differentiation: comparing to the orthogonal direction, detaching in the direction parallel to cell orientation needed higher force and work. The NGF treatment significantly increased relative cell stiffness while it was cell orientation-independent. The immunofluorescent data showed that CoCl2 treatment caused a significant increase of HIF-1α protein and this confirmed that the cell was under hypoxia adaptation.
The cell shape is related to the internal cell structure, our result showed that the similar cell shapes may not correspond to the same mechanical properties. Presently, the cell adhesion model can’t depict the cells with irregular shapes. By analyzing the effects of cell orientations, we further understood the characteristic of cytodetachment and facilitated more applications in the future.

Acker, T. and H. Acker (2004). "Cellular oxygen sensing need in CNS function: physiological and pathological implications." J Exp Biol 207(Pt 18): 3171-88.
Athanasiou, K. A., B. S. Thoma, et al. (1999). "Development of the cytodetachment technique to quantify mechanical adhesiveness of the single cell." Biomaterials 20(23-24): 2405-15.
Bamburg, J. R. (2003). "Introduction to cytoskeletal dynamics and pathfinding of neuronal growth cones." J Histochem Cytochem 51(4): 407-9.
Beaujean, D., C. Rosenbaum, et al. (2003). "Combinatorial code of growth factors and neuropeptides define neuroendocrine differentiation in PC12 cells." Exp Neurol 184(1): 348-58.
Bergeron, M., J. M. Gidday, et al. (2000). "Role of hypoxia-inducible factor-1 in hypoxia-induced ischemic tolerance in neonatal rat brain." Ann Neurol 48(3): 285-96.
Bieberich, E. and G. E. Anthony (2004). "Neuronal differentiation and synapse formation of PC12 and embryonic stem cells on interdigitated microelectrode arrays: contact structures for neuron-to-electrode signal transmission (NEST)." Biosens Bioelectron 19(8): 923-31.
Bito, H., T. Furuyashiki, et al. (2000). "A critical role for a Rho-associated kinase, p160ROCK, in determining axon outgrowth in mammalian CNS neurons." Neuron 26(2): 431-41.
Ceradini, D. J., A. R. Kulkarni, et al. (2004). "Progenitor cell trafficking is regulated by hypoxic gradients through HIF-1 induction of SDF-1." Nat Med 10(8): 858-64.
Connolly, J. L., P. J. Seeley, et al. (1985). "Regulation of growth cone morphology by nerve growth factor: a comparative study by scanning electron microscopy." J Neurosci Res 13(1-2): 183-98.
Corley, K. M., C. J. Taylor, et al. (2005). "Hypoxia-inducible factor 1alpha modulates adhesion, migration, and FAK phosphorylation in vascular smooth muscle cells." J Cell Biochem 96(5): 971-85.
Cowden Dahl, K. D., S. E. Robertson, et al. (2005). "Hypoxia-inducible factor regulates alphavbeta3 integrin cell surface expression." Mol Biol Cell 16(4): 1901-12.
Doornaert, B., V. Leblond, et al. (2003). "Time course of actin cytoskeleton stiffness and matrix adhesion molecules in human bronchial epithelial cell cultures." Exp Cell Res 287(2): 199-208.
Edsjo, A., L. Holmquist, et al. (2007). "Neuroblastoma as an experimental model for neuronal differentiation and hypoxia-induced tumor cell dedifferentiation." Semin Cancer Biol 17(3): 248-56.
Fujita, K., P. Lazarovici, et al. (1989). "Regulation of the differentiation of PC12 pheochromocytoma cells." Environ Health Perspect 80: 127-42.
Gallant, N. D., K. E. Michael, et al. (2005). "Cell adhesion strengthening: contributions of adhesive area, integrin binding, and focal adhesion assembly." Mol Biol Cell 16(9): 4329-40.
Goldberg, D. J. and D. W. Burmeister (1986). "Stages in axon formation: observations of growth of Aplysia axons in culture using video-enhanced contrast-differential interference contrast microscopy." J Cell Biol 103(5): 1921-31.
Greene, L. A. and A. S. Tischler (1976). "Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor." Proc Natl Acad Sci U S A 73(7): 2424-8.
Hall, A. (2005). "Rho GTPases and the control of cell behaviour." Biochem Soc Trans 33(Pt 5): 891-5.
Hammer, D. A. and D. A. Lauffenburger (1987). "A dynamical model for receptor-mediated cell adhesion to surfaces." Biophys J 52(3): 475-87.
Hirose, M., T. Ishizaki, et al. (1998). "Molecular dissection of the Rho-associated protein kinase (p160ROCK)-regulated neurite remodeling in neuroblastoma N1E-115 cells." J Cell Biol 141(7): 1625-36.
Hoben, G., W. Huang, et al. (2002). "Quantification of varying adhesion levels in chondrocytes using the cytodetacher." Ann Biomed Eng 30(5): 703-12.
Hynes, R. O. (1992). "Integrins: versatility, modulation, and signaling in cell adhesion." Cell 69(1): 11-25.
Indovina, P., A. Ferrante, et al. (2006). "Hypoxia and ionizing radiation: changes in adhesive properties and cell adhesion molecule expression in MG-63 three-dimensional tumor spheroids." Cell Commun Adhes 13(3): 185-98.
Isono, F., H. R. Widmer, et al. (1994). "Epidermal growth factor induces PC12 cell differentiation in the presence of the protein kinase inhibitor K-252a." J Neurochem 63(4): 1235-45.
Iyer, N. V., S. W. Leung, et al. (1998). "The human hypoxia-inducible factor 1alpha gene: HIF1A structure and evolutionary conservation." Genomics 52(2): 159-65.
Kaverina, I., O. Krylyshkina, et al. (2002). "Regulation of substrate adhesion dynamics during cell motility." Int J Biochem Cell Biol 34(7): 746-61.
Kotake-Nara, E. and K. Saida (2006). "Endothelin-2/vasoactive intestinal contractor: regulation of expression via reactive oxygen species induced by CoCl2, and Biological activities including neurite outgrowth in PC12 cells." ScientificWorldJournal 6: 176-86.
Kotake-Nara, E. and K. Saida (2007). "Characterization of CoCl2-induced reactive oxygen species (ROS): Inductions of neurite outgrowth and endothelin-2/vasoactive intestinal contractor in PC12 cells by CoCl2 are ROS dependent, but those by MnCl2 are not." Neurosci Lett 422(3): 223-7.
Kotake-Nara, E., S. Takizawa, et al. (2005). "Cobalt chloride induces neurite outgrowth in rat pheochromocytoma PC-12 cells through regulation of endothelin-2/vasoactive intestinal contractor." J Neurosci Res 81(4): 563-71.
Lambrechts, A., V. Jonckheere, et al. (2006). "Profilin-I-ligand interactions influence various aspects of neuronal differentiation." J Cell Sci 119(Pt 8): 1570-8.
Letourneau, P. C. (1975). "Cell-to-substratum adhesion and guidance of axonal elongation." Dev Biol 44(1): 92-101.
Levitan, I. B. and L. K. Kaczmarek. (2002). "The neuron :cell and molecular biology." Oxford University Press 12.
Li, B. S., L. Zhang, et al. (2000). "Integrin alpha(1) beta(1)-mediated activation of cyclin-dependent kinase 5 activity is involved in neurite outgrowth and human neurofilament protein H Lys-Ser-Pro tail domain phosphorylation." J Neurosci 20(16): 6055-62.
Li, S., N. F. Huang, et al. (2005). "Mechanotransduction in endothelial cell migration." J Cell Biochem 96(6): 1110-26.
Lin, W. H., D. Higgins, et al. (1993). "Manganese induces spreading and process outgrowth in rat pheochromocytoma (PC12) cells." J Neurosci Res 34(5): 546-61.
Lotz, M. M., C. A. Burdsal, et al. (1989). "Cell adhesion to fibronectin and tenascin: quantitative measurements of initial binding and subsequent strengthening response." J Cell Biol 109(4 Pt 1): 1795-805.
Mazia, D., G. Schatten, et al. (1975). "Adhesion of cells to surfaces coated with polylysine. Applications to electron microscopy." J Cell Biol 66(1): 198-200.
Mongiu, A. K., E. L. Weitzke, et al. (2007). "Kinetic-structural analysis of neuronal growth cone veil motility." J Cell Sci 120(Pt 6): 1113-25.
Nusser, N., E. Gosmanova, et al. (2002). "Nerve growth factor signals through TrkA, phosphatidylinositol 3-kinase, and Rac1 to inactivate RhoA during the initiation of neuronal differentiation of PC12 cells." J Biol Chem 277(39): 35840-6.
O'Driscoll, C. M. and A. M. Gorman (2005). "Hypoxia induces neurite outgrowth in PC12 cells that is mediated through adenosine A2A receptors." Neuroscience 131(2): 321-9.
Pacary, E., H. Legros, et al. (2006). "Synergistic effects of CoCl(2) and ROCK inhibition on mesenchymal stem cell differentiation into neuron-like cells." J Cell Sci 119(Pt 13): 2667-78.
Pacary, E., E. Tixier, et al. (2007). "Crosstalk between HIF-1 and ROCK pathways in neuronal differentiation of mesenchymal stem cells, neurospheres and in PC12 neurite outgrowth." Mol Cell Neurosci 35(3): 409-23.
Parran, D. K., W. R. Mundy, et al. (2001). "Effects of methylmercury and mercuric chloride on differentiation and cell viability in PC12 cells." Toxicol Sci 59(2): 278-90.
Pollock, J. D., M. Krempin, et al. (1990). "Differential effects of NGF, FGF, EGF, cAMP, and dexamethasone on neurite outgrowth and sodium channel expression in PC12 cells." J Neurosci 10(8): 2626-37.
Prass, K., K. Ruscher, et al. (2002). "Desferrioxamine induces delayed tolerance against cerebral ischemia in vivo and in vitro." J Cereb Blood Flow Metab 22(5): 520-5.
Ronn, L. C., I. Ralets, et al. (2000). "A simple procedure for quantification of neurite outgrowth based on stereological principles." J Neurosci Methods 100(1-2): 25-32.
Saffell, J. L., F. S. Walsh, et al. (1992). "Direct activation of second messenger pathways mimics cell adhesion molecule-dependent neurite outgrowth." J Cell Biol 118(3): 663-70.
Sagvolden, G., I. Giaever, et al. (1999). "Cell adhesion force microscopy." Proc Natl Acad Sci U S A 96(2): 471-6.
Schubert, D. and C. Whitlock (1977). "Alteration of cellular adhesion by nerve growth factor." Proc Natl Acad Sci U S A 74(9): 4055-8.
Schulze, I. and J. R. Perez-Polo (1982). "Nerve growth factor and cyclic AMP: opposite effects on neuroblastoma-substrate adhesion." J Neurosci Res 8(2-3): 393-411.
Studer, L., M. Csete, et al. (2000). "Enhanced proliferation, survival, and dopaminergic differentiation of CNS precursors in lowered oxygen." J Neurosci 20(19): 7377-83.
Suter, D. M. and P. Forscher (2001). "Transmission of growth cone traction force through apCAM-cytoskeletal linkages is regulated by Src family tyrosine kinase activity." J Cell Biol 155(3): 427-38.
Tawil, N., P. Wilson, et al. (1993). "Integrins in point contacts mediate cell spreading: factors that regulate integrin accumulation in point contacts vs. focal contacts." J Cell Biol 120(1): 261-71.
Tischler, A. S. and L. A. Greene (1978). "Morphologic and cytochemical properties of a clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor." Lab Invest 39(2): 77-89.
Tomita, S., M. Ueno, et al. (2003). "Defective brain development in mice lacking the Hif-1alpha gene in neural cells." Mol Cell Biol 23(19): 6739-49.
Wang, G., T. K. Hazra, et al. (2000). "Mitochondrial DNA damage and a hypoxic response are induced by CoCl(2) in rat neuronal PC12 cells." Nucleic Acids Res 28(10): 2135-40.
Wang, G. L. and G. L. Semenza (1993). "Desferrioxamine induces erythropoietin gene expression and hypoxia-inducible factor 1 DNA-binding activity: implications for models of hypoxia signal transduction." Blood 82(12): 3610-5.
Wiener, C. M., G. Booth, et al. (1996). "In vivo expression of mRNAs encoding hypoxia-inducible factor 1." Biochem Biophys Res Commun 225(2): 485-8.
Wu, C. C., H. W. Su, et al. (2005). "Quantitative measurement of changes in adhesion force involving focal adhesion kinase during cell attachment, spread, and migration." Biochem Biophys Res Commun 329(1): 256-65.
Yamamoto, A., S. Mishima, et al. (1998). "A new technique for direct measurement of the shear force necessary to detach a cell from a material." Biomaterials 19(7-9): 871-9.
Yamamoto, A., S. Mishima, et al. (2000). "Quantitative evaluation of cell attachment to glass, polystyrene, and fibronectin- or collagen-coated polystyrene by measurement of cell adhesive shear force and cell detachment energy." J Biomed Mater Res 50(2): 114-24.
Yoo, Y. E., J. H. Hong, et al. (2004). "Iron enhances NGF-induced neurite outgrowth in PC12 cells." Mol Cells 17(2): 340-6.
Zhu, J., Y. S. Wang, et al. (2008). "Focal adhesion kinase signaling pathway participates in the formation of choroidal neovascularization and regulates the proliferation and migration of choroidal microvascular endothelial cells by acting through HIF-1 and VEGF expression in RPE cells." Exp Eye Res.
Zou, J., R. A. Rabin, et al. (1993). "Ethanol enhances neurite outgrowth in primary cultures of rat cerebellar macroneurons." Brain Res Dev Brain Res 72(1): 75-84.